Abcb5(+) stem cells for treating ocular disease

ABSTRACT

Various aspects and embodiments of the present invention are directed to methods of treating a subject having an ocular condition, methods of isolating ocular stem cells, methods of selecting and/or producing ocular grafts for transplantation, and methods of promoting ocular cell regeneration as well as to grafts and preparations containing isolated ocular stem cells characterized by the expression of ABCB5 on their cell surface.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application No. 61/766,424, filed Feb. 19, 2013, which isincorporated by reference herein in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under 5R01CA113796awarded by the National Institutes of Health and the National CancerInstitute. The government has certain rights in the invention.

BACKGROUND OF INVENTION

Limbal stem cells have been identified as slow-cycling, label-retainingcells in mice. Limbal stem cells express the nuclear transcriptionfactor ΔNp63α in humans and lack expression of corneal epithelialdifferentiation markers such as KRT12 [2,8] (FIG. 1A). Limbal stem cellsgenerate transient amplifying cells, which express the eye developmentmaster regulator PAX6 [9] and, during corneal development andregeneration, migrate out of the limbus to give rise to the KRT12(+)central corneal epithelium [10].

SUMMARY OF INVENTION

The present invention, in some aspects, is directed generally to the useof ABCB5(+) stem cells (e.g., human stem cells) for the treatment ofocular conditions such as, for example, corneal diseases and/or retinaldiseases. The invention is based, in part, on the discovery that ABCB5is expressed in stem cells of the eye, such ABCB5(+) stem cells arerequired for normal eye development, and when administered to subjectshaving ocular wounds (e.g., ocular surface wounds), these cells arecapable of cell regeneration. For example, ABCB5(+) limbal stem cells,required for normal corneal development, are capable of cornealregeneration. Similarly, ABCB5(+) retinal pigment epithelium (RPE)cells, required for normal retinal development, are capable of retinalregeneration.

Thus, in some aspects of the invention, provided herein are methods oftreating a subject having an ocular condition, comprising administeringto the subject isolated ABCB5(+) stem cells in an amount effective toregenerate ocular cells in the subject.

In some embodiments, the ocular condition is a corneal disease. In someembodiments, the corneal disease is blindness due to limbal stem celldeficiency (LSCD). “Limbal stem cell deficiency” herein refers to severeor total, unilateral or partial LSCD [5]. In some embodiments, isolatedABCB5(+) limbal stem cells are administered to a subject to treat acorneal disease.

In some embodiments, the ocular condition is a retinal disease. In someembodiments, the retinal disease is macular degeneration. In someembodiments, the retinal disease is retinitis. In some embodiments,isolated ABCB5(+) retinal stem cells (e.g., ABCB5(+) RPE stem cells) areadministered to a subject to treat a corneal disease.

In some embodiments, the ocular condition is an ocular wound.

In some embodiments, the isolated ABCB5(+) stem cells are administeredas an ocular graft. In some embodiments, the ocular grafts contain oneto about 10⁷ isolated ABCB5(+) stem cells. In some embodiments, morethan 10⁷ isolated ABCB5(+) stem cells may be administered as an oculargraft.

In other aspects of the invention, provided herein are methods ofisolating limbal stem cells from a mixed population of ocular cells, themethods comprising providing a mixed population of ocular cells andisolating ABCB5(+) limbal stem cells from the mixed population.

In yet other aspects of the invention, provided herein are methods ofidentifying the number of ABCB5(+) limbal stem cells in the oculargraft, comparing the number of ABCB5(+) limbal stem cells to the totalcell population of the graft, and based on the comparison, selecting theocular graft for transplantation.

In some embodiments, the methods comprise contacting cells of the mixedpopulation with an antibody that selectively binds to human ABCB5.

In still other aspects of the invention, provided herein are methods ofproducing ocular grafts for transplantation to a subject, the methodscomprising seeding a substrate with isolated ABCB5(+) stem cells toproduce the ocular graft.

In some embodiments, the substrate comprises fibrin gel, amnioticmembrane, aminoglycans, or a combination thereof. In some embodiments,the substrate is an artificial cornea. In such embodiments, thesubstrate, for example, an artificial cornea, comprises acellularcollagen.

In some aspects of the invention, provided herein are ocular graftsenriched with isolated ABCB5(+) stem cells for transplantation in asubject.

In still other aspects of the invention, provided herein are methods ofpromoting ocular cell regeneration, comprising identifying limbal stemcells as ABCB5(+) limbal stem cells and administering to a subject inneed thereof the ABCB5(+) limbal stem cells in an amount effective topromote ocular cell regeneration.

In further aspects of the invention, provided herein are isolatedpreparations of limbal stem cells characterized by the expression ofABCB5 on the cell surface.

In some embodiments, the isolated ABCB5(+) limbal stem cells areadministered as an ocular graft.

In some embodiments, the subject is administered one to about 10⁷isolated ABCB5(+) limbal stem cells by grafting.

In some embodiments, the isolated ABCB5(+) stem cells are isolatedABCB5(+) human stem cells.

In some embodiments, the isolated ABCB5(+) stem cells are allogeneicstem cells. In some embodiments, the isolated ABCB5(+) stem cells aresyngeneic stem cells.

In some embodiments, the isolated ABCB5(+) stem cells are ABCB5(+)ocular stem cells. In some embodiments, the isolated ABCB5(+) ocularstem cells are isolated ABCB5(+) limbal stem cells. In some embodiments,the isolated ABCB5(+) limbal stem cells are isolated ABCB5(+) humanlimbal stem cells.

In some embodiments, the isolated ABCB5(+) stem cells are not skin stemcells (e.g., mesenchymal stem cells).

In some embodiments, the isolated ABCB5(+) stem cells are expandedex-vivo prior to the administering step.

In some embodiments, the subject is a mammal. In some embodiments, themammal is a human.

In some aspects of the invention, provided herein are kits that includea container housing any of the foregoing grafts or stem cellpreparations and instructions for administering the graft or preparationto a subject in need thereof.

Use of a graft or stem cell preparation of the invention for treating anocular condition is also provided as an aspect of the invention.

A method for manufacturing a medicament of a stem cell preparation ofthe invention for treating an ocular condition is also provided.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Each of the above embodiments and aspects may belinked to any other embodiment or aspect. Also, the phraseology andterminology used herein is for the purpose of description and should notbe regarded as limiting. The use of “including,” “comprising,” or“having,” “containing,” “involving,” and variations thereof herein, ismeant to encompass the items listed thereafter and equivalents thereofas well as additional items.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1A shows a schematic illustration of corneal structures and thelimbal stem cell niche.

FIG. 1B shows representative flow cytometric analyses of BrdU-labeleddissociated murine corneal cells identifying the presence of alabel-retaining cell population in the limbus.

FIG. 1C shows immunofluorescence images depicting co-expression of ABCB5and BrdU in murine limbus.

FIG. 1D shows representative flow cytometric analyses depictingco-expression of ABCB5 and BrdU in murine limbus. Bar graph (right)illustrates quantitative analysis of independent experiments (n=4).

FIG. 1E shows representative immunohistochemical analyses of tangentiallimbal cross-sections from human corneas depicting ABCB5 expression(green) in the basal epithelial layer.

FIG. 1F shows representative immunohistochemical analyses of tangentiallimbal cross-sections from human corneas depicting co-expression ofABCB5 (red) with ΔNp63α (green).

FIG. 1G shows representative cytometric analyses of human limbalepithelial cells depicting co-expression of ABCB5 with ΔNp63α. Bargraphs show ΔNp63α expression on ABCB5(+) and ABCB5(−) cells (leftpanel), and ABCB5 expression on ΔNp63α(+) and ΔNp63α(−) cells (rightpanel). Data are depicted as mean±s.e.m., n=3 experiments.

FIG. 1H shows dual color flow cytometry analyses of ABCB5 and KRT12co-expression.

FIG. 1I shows representative immunohistochemical analyses of ABCB5expression in limbal biopsies from patients with limbal stem celldeficiency (LSCD) performed at the time of surgery and from theirrespective donors. Bar graphs show the number of ABCB5(+) cells (green)in healthy donors and patients with LSCD (n=8 sections perpatient/donor).

FIG. 2A shows a schematic of the murine Abcb5 gene locus and proteintopology. The topological structure was determined by the TMHMM membranetopology prediction algorithm and displayed using TOPO2 software. Aminoacid residues deleted in Abcb5 knockout (KO) (mutant) mice arehighlighted in red.

FIG. 2B shows a schematic summary of the strategy employed forgeneration of the Abcb5 KO mouse.

FIG. 2C, left panel, shows electrophoresis images of a polymerase chainreaction (PCR) analysis of the genomic DNA used for mouse genotyping,demonstrating a 113-base pair wild type (WT) allele and a 322-base pairdeleted allele. FIG. 2C, right panel, shows Western blots of murineprotein lysates with ABCB5 monoclonal antibody (mAb) 3C2-1D12, whichrevealed loss of a 80 kD protein band of predicted size in Abcb5 KOmice.

FIG. 2D shows images of a phenotypic characterization of murine Abcb5 WTand Abcb5 KO corneas using slit lamp examination (left panels),hematoxylin and eosin (H&E) staining (middle panels) and4′,6-diamidino-2-phenylindole (DAPI) staining (right panels). Bar graphsbelow depict the number of DAPI(+) epithelial cells in Abcb5 KO andAbcb5 WT murine central cornea and limbus. Data shown representmeans±s.e.m., n=4 experiments.

FIG. 2E shows LC-biotin diffusion analyses and immunofluorescenceprotein expression analyses of PAX6, KRT12 and KRT14 in Abcb5 WT andAbcb5 KO mice. Bar graphs depict percent PAX6(+) and KRT12(+) epithelialcells in Abcb5 KO and Abcb5 WT mice. Data shown represent means±s.e.m.,n=6 experiments.

FIG. 2F shows H&E and DAPI staining of Abcb5 WT and Abcb5 KO corneas 48hours after epithelial debridement wounding. Bar graph (bottom)represents the number of DAPI(+) cells per section in Abcb5 WT and Abcb5KO mice. Data shown represent means±s.e.m., n=4 experiments.

FIG. 2G shows immunofluorescence analyses of Ki67 in the limbus andcentral cornea of Abcb5 WT and Abcb5 KO mice 48 hours after epithelialdebridement wounding. Bar graphs (bottom) represent the percentage ofKi67(+) in limbus and in cornea Abcb5 KO and Abcb5 WT mice(means±s.e.m., n=4 experiments, respectively).

FIG. 2H shows immunofluorescence analyses of TUNEL staining in thelimbus and central cornea of Abcb5 WT and Abcb5 KO mice 48 hours afterepithelial debridement wounding. Bar graphs (bottom) represent thepercentage of TUNEL+epithelial cells in limbus and in cornea in Abcb5 KOand Abcb5 WT mice (means±s.e.m., n=4 experiments, respectively).

FIG. 3A shows flow cytometry analyses showing loss of BrdUlabel-retaining cells in Abcb5 KO and Abcb5 WT limbal epithelial cellsafter an 8-week chase.

FIG. 3B shows flow cytometry analyses showing loss of BrdUlabel-retaining cells in Abcb5 KO and Abcb5 WT limbal epithelial cellsafter a 1-week chase (means±s.e.m., n=6 experiments).

FIG. 3C shows immunofluorescence analyses of Ki67 expression in Abcb5 WTand Abcb5 KO mouse limbus and cornea. Bar graphs on the right illustratethe percentages of Ki67(+) cells in Abcb5 WT and Abcb5 KO mice in thelimbus and cornea. Illustrated are means±s.e.m. (n=3 experiments).

FIG. 3D shows a graph of mRNA expression of p53, p63, p21 and p16 inAbcb5 WT and Abcb5 KO corneas. Bars represent relative mRNA expressionlevels in Abcb5 KO mice as a percentage of mRNA expression levels inAbcb5 WT mice (means±s.e.m., n=4 experiments).

FIG. 3E shows a schematic summary of the role of ABCB5 in cell cycleregulation and normal corneal development and regeneration. Abrogationof ABCB5 expression in Abcb5 KO mice (blue) results in loss of BrdU(+)label-retaining cells and down regulation of critical cell cycleregulators, including p63. This leads to increased cellularproliferation as evidenced by enhanced Ki67 expression in Abcb5 KO mice.Augmented proliferation and inability to withdraw from the cell cycleexplain the profound differentiation deficiencies, evidenced bydecreased PAX6 and KRT12 expression and increased rates of apoptosis inAbcb5 KO mice, evidenced by enhanced TUNEL staining.

FIG. 4A shows analyses of murine syngeneic donor cell transplantsgrafted onto C57BL/6 recipient mice.

FIG. 4B shows analyses of human xenogeneic donor cell transplantsgrafted onto immunodeficient NSG recipient mice. The images show tissuefive weeks post transplantation performed for the treatment ofexperimentally induced LSCD. In FIGS. 4A and 4B, recipient mice receivedfibrin gel grafts containing no donor cells (rows 2, respectively),ABCB5(−) cells (rows 3, respectively), unsegregated limbal epithelialcells (rows 4, respectively), or ABCB5(+) cells (rows 5, respectively).As a reference, normal untreated (without induced LSCD) C57BL/6 and NSGmurine corneas are shown in rows 1 of FIG. 4A and FIG. 4B, respectively.Corneal transparency was evaluated by slit lamp examination (FIG. 4A,4B, columns 1). Epithelial integrity and regeneration were evaluated byH&E staining (columns 2—20× magnification; columns 3—40× magnification)for epithelial thickness and stratification, by periodic acid-Schiffstaining (PAS) for detection Goblet cells associated withneovascularization (FIG. 4A, column 4), and Krt12 staining (green) fordetection of differentiated corneal epithelial cells (FIG. 4A, 4B,columns 5). Nuclei are stained with DAPI (red). Bar graphs on the rightshow the percentages of murine KRT12(+) cells (FIG. 4A) or humanKRT12(+) cells (FIG. 4B) in recipient corneas 5 weeks aftertransplantation. The right lower panel in (FIG. 4B) shows RT-PCRanalyses of murine eyes transplanted with human cells for evaluation ofhuman donor cell contribution to corneal repair.

FIG. 5A shows a schematic summary of the experimental design for BrdUpulse-chase experiments.

FIG. 5B shows representative flow cytometric analyses depicting specificstaining of BrdU label-retaining cells in limbal epithelial cells of WTmice that did not receive BrdU (left two panels) or WT mice thatreceived BrdU followed by an 8 week chase (right two panels). Limbalepithelial cells were recovered and stained with either anti-BrdUantibody (Ab), or with an isotype control Ab. The percentages ofBrdU-positive cells within the gate are indicated on each plot.

FIG. 6 shows a schematic illustration of tangential limbal crosssections from human donor corneas, indicating the location of the limbalepithelium. ABCB5(+) cells (schematically depicted as green coloredcells) were found located in the basal epithelial layer.

FIG. 7 shows limbal biopsies from a patient with LSCD (patient 1).Limbal biopsies were obtained from patient 1 with a chemical burn priorto receiving a penetrating keratoplasty plus kerato-limbal allograftfrom a cadaveric donor eye (donor 1). Serial cross sections of thebiopsies were stained with either H&E, isotype control Ab or ABCB5 mAb.ABCB5 staining in the limbal epithelium of donor 1 revealed nests ofABCB5-positive cells, whereas ABCB5 positivity was reduced in the limbalepithelium of patient 1. Photographs of immunofluorescent staining aremontages of sequential photos at 20× magnification.

FIG. 8 shows limbal biopsies from a patient with LSCD (patient 2).Limbal biopsies were obtained from patient 2 with an autoimmune cornealmelt, peripheral ulcerative keratitis and partial limbal stem celldeficiency prior to receiving a kerato-limbal autograft from thepatient's normal contralateral eye (donor 2). Serial sections of thebiopsies were stained with either H&E, isotype control Ab or ABCB5 mAb.ABCB5 positivity was present in the basal layer of the limbal epitheliumof donor 2, while a dramatically reduced epithelial layer and no ABCB5staining were observed in the limbus of patient 2. Photographs ofimmunofluorescent staining are montages of sequential photos at 20×magnification.

FIG. 9 shows representative flow cytometry analyses of either the limbalor the central corneal epithelium of Abcb5 WT and Abcb5 KO mice. Forwardscatter (FSC) and Side scatter (SSC) indicates cellular size andgranularity, respectively. Central corneal epithelium of Abcb5 KO miceshowed a reduced number of epithelial cells compared to Abcb5 WTepithelium (left panels), caused by a reduction in larger cells (rightgates), but not smaller cells (left gates). There was no reduction inthe number of limbal epithelial cells (right panels). Representativeresults of samples pooled from four eyes are shown (n=3 experiments).

FIG. 10 shows representative flow cytometry analyses of epithelial cellsharvested from either the limbus (top) or the central cornea (bottom) ofAbcb5 WT and Abcb5 KO mice. Recovered cells were stained with isotypecontrol antibody, anti-Pax6 antibody or anti-Krt12 antibody. There was areduced frequency of PAX6(+) and KRT12(+) epithelial cells in thecentral cornea of Abcb5 KO mice and a corresponding reduced frequency ofPAX6(+) cells in the limbus of Abcb5 KO mice. Red gates identify PAX6(+)or KRT12(+) cells compared to isotype control staining. Representativeanalyses of n=3 experiments are shown.

FIG. 11A shows a wound area to be debrided marked with a 2 mm trephineand the epithelium removed.

FIG. 11B shows a DAPI-stained cross section of the cornea immediatelyfollowing central epithelial debridement depicting the wound margins andexposed central corneal stroma. Image is a montage of sequential photosat 10× magnification.

FIG. 11C shows fluorescent images of corneal epithelial wound closuremonitoring at 1, 24, and 48 hours post debridement.

FIG. 11D shows a graph of wound closure rates, which were notsignificantly different between Abcb5 WT and Abcb5 KO mice (summary ofn=2 replicate experiments).

FIG. 12 shows representative DAPI-stained composite corneal crosssections of Abcb5 WT (top) and Abcb5 KO (bottom) mice 48 hours after acorneal epithelial debridement wound, demonstrating a reduced number ofepithelial cells in Abcb5 KO mice. The white dashed line demarcates theepithelium from stroma; the white box indicates area shown at 20×magnification (montage pictures are at 10× magnification); white linesdemarcate the area in which epithelial cells were counted. Epithelialcells were counted within the standardized area in at least threeconsecutive composite cross sections in three replicate mice per groupin two separate experiments (data shown in FIG. 2F).

FIG. 13 shows representative TUNEL-stained composite corneal crosssections of Abcb5 WT (top) and Abcb5 KO (bottom) mice 48 hours after acorneal epithelial debridement wound, demonstrating increased numbers ofapoptotic cells in Abcb5 KO mice. Areas defined by the white box areshown at 20× magnification (montage pictures at 10× magnification). Thenumber of TUNEL-positive epithelial cells was counted, and the data fromtwo replicate experiments are summarized in FIG. 2H.

FIG. 14A shows a schematic illustration of the recovery and separationof ABCB5(+) and ABCB5(−) limbal epithelial cells from donor corneasfollowed by preparation of fibrin gels containing donor cells.

FIG. 14B shows a schematic illustration of induction of limbal stem celldeficiency in recipient mice and transplantation of donor grafts.

FIG. 15A shows representative flow cytometry analyses showing sortinggates and viability of murine donor limbal epithelial cells. Viabilityis shown as the percentage of cells excluding DAPI.

FIG. 15B shows post-sort analyses depicting the purity and viability ofABCB5(+)-enriched and ABCB5(−)-enriched subpopulations of limbalepithelial cells isolated from murine donors. Viability is shown as thepercentage of cells excluding DAPI.

FIG. 15C shows representative flow cytometry analyses showing sortinggates and viability of human donor limbal epithelial cells.

FIG. 15D shows post-sort analyses depicting the purity and viability ofABCB5(+)-enriched and ABCB5(−)-enriched subpopulations of limbalepithelial cells isolated from human donors. Viability is shown as thepercentage of cells excluding DAPI.

FIG. 16 shows representative H&E composite corneal cross sections ofrecipient C57BL/6J mice 5 weeks after receiving an induced limbal stemcell deficiency (LSCD) followed by engraftment of donor fibrin geltransplants containing the following syngeneic murine limbal epithelialcell subpopulations: (i) no cells (negative control), (ii) ABCB5(+)cells, (iii) ABCB5(−) cells or (iv) unsegregated cells. A normaluntreated cornea (no LSCD) served as a positive control. The positivecontrol displays the typical stratified corneal epithelium andiridocorneal angle. Mice receiving transplants with no cells displayedthe typical conjunctivalization that occurs following a LSCD, i.e.,unstratified conjunctival epithelium covers the cornea with extensiveinflammation, neovascularization, and stromal edema. Synechia (where theiris adheres to the cornea) is typical of intense anterior segmentinflammation. In contrast, mice that received transplants of ABCB5(+)cells, but not ABCB5(−) cells, displayed a restored stratified cornealepithelium with no evidence of inflammation, neovascularization, stromaledema, or synechia. Mice that received transplants of unsegregatedlimbal epithelial cells displayed areas of stromal edema withunstratified epithelium, while other parts of the cornea containednormal stratified epithelial cells.

FIG. 17 shows representative H&E composite corneal cross sections ofrecipient immunodeficient NSG mice 5 weeks after LSCD induction followedby transplantation of donor fibrin gel grafts containing the followinghuman limbal epithelial cell subpopulations: (i) no cells (negativecontrol), (ii) ABCB5(+) cells, (iii) ABCB5(−) cells, and (iv)unsegregated cells. A normal untreated NSG cornea (no LSCD) served as apositive control. The positive control displays the typical stratifiedcorneal epithelium and iridocorneal angle. Mice that receivedtransplants with no cells displayed evidence of conjunctivalization thatoccurs following a LSC deficiency, i.e., unstratified conjunctivalepithelium covers the cornea with extensive neovascularization andsynechia (anterior segment inflammation is muted in NSG mice due totheir immunodeficiency). In contrast, mice that received transplantscontaining ABCB5(+) cells displayed areas of restored stratifiedepithelium, whereas mice that received ABCB5(−) cell grafts did not.

FIG. 18 shows representative immunofluorescent Krt12 staining (green) ofrecipient C57BL/6J mice 5 weeks after an LSCD induction followed bytransplantation of donor fibrin gel grafts containing the followingsyngeneic murine limbal epithelial cell subpopulations: (i) no cells(negative control), (ii) ABCB5(+) cells, (iii) ABCB5(−) cells, or (iv)unsegregated cells. Normal untreated murine cornea (no LSCD), shown hereas a positive control, displayed high intensity of KRT12 staining. Asexpected, mice that received grafts containing no cells, displayed noKRT12 expression. In contrast, mice transplanted with ABCB5(+) cells,exhibited significantly enhanced KRT12 expression in comparison to micetransplanted with unsegregated limbal epithelial cells. No KRT12expression was detected in mice transplanted with ABCB5(−) cells. Thewhite box depicts the area shown at 40× magnification. Montage imagesare shown at 10× magnification.

DETAILED DESCRIPTION

Corneal epithelial homeostasis and regeneration are sustained by apopulation of limbal stem cells (LSCs) residing in the basal limbalepithelium of the eye [1-3]. These cells generate new corneal cells toreplace damaged ones, and loss of LSCs due to injury or disease is amajor cause of blindness worldwide [4]. Transplantation of LSCs from ahealthy eye is often the only therapeutic option available to patientswith LSCD. Transplant success depends foremost on the frequency of LSCswithin grafts [5]. However, prior to the present invention, a limbalstem cell gene that permits prospective enrichment of this cell subsethad not been reported [5].

The present invention is based, in part, on the findings thatATP-binding cassette, sub-family B (MDR/TAP), member 5 (ABCB5) [6,7]marks LSCs and is required for limbal stem cell maintenance, cornealdevelopment and repair, and that ABCB5-positive (ABCB5 (+)) LSCsprospectively isolated from donors possess the exclusive capacity torestore the cornea upon grafting. Thus, various aspects and embodimentsof the invention are directed to methods of treating a subject having anocular condition, methods of isolating ABCB5(+) stem cells of the eye,methods of selecting and/or producing ocular grafts for transplantation,and methods of promoting ocular cell regeneration as well as to graftsand preparations containing isolated ocular stem cells characterized bythe expression of ABCB5 on their cell surface.

The inventors of the present invention demonstrate herein that ABCB5 isuniformly expressed on in vivo label-retaining LSCs in wild type miceand on ΔNp63α-positive LSCs in healthy humans. Consistent with thesefindings, the inventors also demonstrate that ABCB5-positive limbal stemcell frequency is significantly reduced in LSCD patients. ABCB5 loss offunction studies using newly generated Abcb5 knockout (KO) mice causeddepletion of quiescent LSCs due to enhanced proliferation and apoptosisand resulted in defective corneal differentiation and wound healing,which explains the demonstrated capacity of ABCB5(+) LSCs to restore thecornea. Results from murine gene KO, in vivo limbal stem cell tracingand limbal stem cell transplantation models, and concurrent findings inphenotypic and functional transplant analyses of human biopsy specimens,provide converging lines of evidence that ABCB5 identifies mammalianLSCs. Identification and prospective isolation of molecularly definedLSCs with essential functions in corneal development and repair hasimportant implications for the treatment of corneal disease,particularly corneal blindness due to LSCD.

“ABCB5(+) stem cells,” as used herein, refers to cells having thecapacity to self-renew and to differentiate into mature cells ofmultiple adult cell lineages. These cells are characterized by theexpression of ABCB5 on the cell surface. In some embodiments of theinvention, ABCB5(+) stem cells are limbal stem cells. In someembodiments of the invention, ABCB5(+) stem cells are retinal stemcells. ABCB5(+) stem cells may be obtained from (e.g., isolated from orderived from) the basal limbal epithelium of the eye or from the retinalpigment epithelium (RPE). In some embodiments, ABCB5(+) stem cells areobtained from human eye. Other ABCB5(+) stem cell types such as, forexample, those obtained from the central cornea may be used in variousaspects and embodiments of the invention.

ABCB5(+) ocular stem cells may be obtained from a subject by isolating asample of eye tissue, including ocular cells of the basal limbalepithelium or RPE, and then purifying the ABCB5(+) stem cells. It willbe apparent to those of ordinary skill in the art that a sample can beenriched for ocular stems cells having ABCB5 in a number of ways. Forexample, ocular stems cells can be selected for through binding of ABCB5on cell surface molecules with antibodies or other binding molecules.Ocular cells may be obtained directly from a donor or retrieved fromcryopreservative storage. The ocular stems cells may, for instance, beisolated using antibodies against ABCB5 and maintained in culture usingstandard methodology or frozen, e.g., in liquid nitrogen, for later use.A non-limiting example of a method that may be used in accordance withthe invention to obtain cells from the eye is described in the Examplessection and is depicted in FIG. 14A.

The present invention contemplates any suitable method of employingABCB5-binding molecules such as, for example, monoclonal antibodies,polyclonal antibodies, human antibodies, chimeric antibodies, humanizedantibodies, single-chain antibodies, F(ab′)2, Fab, Fd, Fv orsingle-chain Fv fragments to separate ABCB5(+) stem cells from a mixedpopulation of ocular cells. Accordingly, included in the presentinvention is a method of producing a population of ABCB5(+) stem cellscomprising the steps of providing a cell suspension of ocular cells;contacting the cell suspension with a monoclonal antibody, or acombination of monoclonal antibodies, which recognize(s) an epitope,including ABCB5, on the ABCB5(+) LSCs; and separating and recoveringfrom the cell suspension the cells bound by the monoclonal antibodies.The monoclonal antibodies may be linked to a solid-phase and utilized tocapture limbal stem cells from eye tissue samples. The bound cells maythen be separated from the solid phase by known methods depending on thenature of the antibody and solid phase.

“Monoclonal antibody,” as used herein, refers to an antibody obtainedfrom a single clonal population of immunoglobulins that bind to the sameepitope of an antigen. Monoclonal based systems appropriate forpreparing cell populations of the invention include magneticbead/paramagnetic particle column utilizing antibodies for eitherpositive or negative selection; separation based on biotin orstreptavidin affinity; and high speed flow cytometric sorting ofimmunofluorescent-stained LSCs mixed in a suspension of other cells.Thus, the methods of the present invention include the isolation of apopulation of LSCs and enhancement using monoclonal antibodies raisedagainst surface antigen ABCB5 (e.g., monoclonal antibodies thatselectively bind ABCB5). In some instances, commercially availableantibodies or antibody fragments that selectively bind ABCB5 may be usedin the methods disclosed herein. Such antibodies are considered toselectively bind to ABCB5 if they bind or are capable of binding toABCB5 with a greater affinity that the affinity with which themonoclonal antibodies may bind to other antigens (i.e., antigens otherthan ABCB5). Such binding may be measured or determined by standardprotein-protein interaction assays (e.g., antibody-antigen orligand-receptor assays) such as, for example, competitive assays,saturation assays or standard immunoassays including, withoutlimitation, enzyme-linked immunosorbent assays, radioimmunoassays andradio-immuno-filter binding assays.

The ABCB5(+) stem cells (e.g., ABCB5(+) LSCs) may be isolated. An“isolated ABCB5(+)stem cell,” as used herein, refers to a cell that hasbeen removed from an organism in which it was originally found, or adescendant of such a cell. An isolated cell also refers to a cell thatis placed into conditions other than the natural environment. Such acell may later be introduced into a second organism or re-introducedinto the organism from which it (or the cell or population of cells fromwhich it descended) was isolated. Such a cell, once manipulatedaccording to the methods of the invention is still considered to be anisolated cell. The term “isolated” does not preclude the later use ofthe cell thereafter in combinations or mixtures with other cells or inan in vivo environment.

“Compositions,” herein, may refer to an isolated cell preparations orgrafts, including tissue grafts and artificial grafts (e.g., acellularcollagen grafts). The compositions of the invention, in some instances,are enriched with isolated ABCB5(+) stem cells. A composition isconsidered to be enriched with isolated ABCB5(+) stem cells if theABCB5(+) stem cells are the predominant cell subtype present in thepreparation. For example, an ABCB5(+) stem cell-enriched composition isa composition in which at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99% or 100% of the cellsof the composition are ABCB5(+) stem cells (e.g., ABCB5(+) LSCs). Insome embodiments, a composition enriched with isolated ABCB5(+) stemcells is one in which less than 50%, less than 45%, less than 40%, lessthan 35%, less than 30%, less than 25%, less than 20%, less than 15%,less than 10%, less than 9%, less than 8%, less than 7%, less than 6%,less than 5%, less than 4%, less than 3%, less than 2% or less than 1%of the cells of the composition are ABCB5(−) cells. In some embodiments,the cells of a composition are only ocular cells. That is, in someembodiments, a composition may not contain non-ocular cells. In someembodiments, a composition may not contain ABCB5(−) cells.

The ABCB5(+) stem cells (e.g., ABCB5(+) LSCs) may be prepared assubstantially pure preparations. The term “substantially pure,” as usedherein, refers to a preparation that is substantially free of cellsother than ABCB5(+) stem cells (e.g., ABCB5(+) LSCs). For example, asubstantially pure preparation of ABCB5(+) stem cells may constitute apreparation in which at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% percent of the total cells present in apreparation are ABCB5(+) stem cells (e.g., ABCB5(+) LSCs).

In some embodiments, isolated and/or substantially pure ABCB5(+) cellpreparations may be packaged in a finished pharmaceutical container suchas an injection vial, ampoule, or infusion bag along with any othercomponents that may be desired, e.g., agents for preserving cells orreducing bacterial growth. The cell preparation may be in unit dosageform.

The ABCB5(+) stem cells (e.g., ABCB5(+) LSCs) are useful for treatingocular conditions. In some embodiments, the ocular condition is anocular wound, which may lead to ocular scarring, which in turn may causedecreased vision or blindness. In some embodiments, the ABCB5(+) stemcells (e.g., ABCB5(+) LSCs) may be used to treat corneal diseases suchas, for example, blindness due to limbal stem cell deficiency (LSCD). Insome embodiments, the ABCB5(+) stem cells (e.g., ABCB5(+) LSCs and/orABCB5(+) RPE stem cells) may be used to treat retinal diseases such as,for example, macular degeneration or retinitis/retinitis pigmentosa.Macular degeneration refers to a group of conditions that includes adeterioration of the macula causing a loss of central vision needed forsharp, clear eyesight. It is a leading cause of vision loss andblindness in those 65 years of age and older. Macular degeneration mayalso be referred to as AMD or ARMD (age-related macular degeneration).Retinitis refers to inflammation of the retina, which may lead toblindness. Retinitis pigmentosa, which may be the result of a geneticcondition or an inflammatory response, refers to a group of inheriteddisorders characterized by progressive peripheral vision loss and nightvision difficulties (nyctalopia) that can lead to central vision loss.

The isolated ABCB5(+) stem cells (e.g., ABCB5(+) LSCS and/or ABCB5(+)RPE stem cells) may be administered to a subject in need thereof in anamount effective to regenerate ocular cells in the subject (referred toherein as an “effective amount” of ABCB5(+) stem cells). In someembodiments, one to about 10⁷ ABCB5(+) stem cells are administered to asubject. In some embodiments, a single isolated ABCB5(+) stem cell isadministered to a subject. In some embodiments, about 10¹ to about 10⁷,about 10¹ to about 10⁶, about 10¹ to about 10⁵, about 10¹ to about 10⁴,about 10¹ to about 10³, about 10¹ to about 10² isolated ABCB5(+) stemcells are administered to a subject. In some embodiments, about 10¹,10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ or more isolated ABCB5(+) stem cells areadministered to a subject. In some embodiments, less than about 10¹isolated ABCB5(+) stem cells are administered to a subject.

In some embodiments, the isolated ABCB5(+) stem cells (e.g., as acomposition in the form of an ABCB5(+) stem cell preparation or graft)may be administered to a subject more than once. Thus, in someembodiments, a subject may be administered multiple doses or grafts(e.g., 2, 3, 4 or more) of isolated ABCB5(+) stem cells over the courseof several weeks, months or years. In some embodiments, the stem cellsare administered again 3 months, 6 months, 9 months, 12 months, 18months, 21 months or 24 months after the first application. The numberof applications and frequency of application may depend, for example, onthe degree of cellular regeneration achieved after the first stem celladministration/transplantation. The number and frequency of stem cellapplications may be determined by a medical professional (e.g., surgeon,physician).

In some embodiments, a subject having an ocular condition has an ocularwound (e.g., dead, damaged or infected ocular cells) in, for example,the corneal epithelium. Thus, the corneal epithelium may be wounded in asubject having an ocular condition in accordance with the invention. Ithas been discovered that ABCB5(+) limbal stem cell grafts can be used torestore the cornea. Thus, in some embodiments, the integrity of thecorneal epithelial surface of the subject is restored followingadministration of an effective amount of ABCB5(+) LSCs. Cornealregeneration may be assessed based on, for example, corneal transparency(e.g., development of clear, rather than opaque, cornea) and/or visualacuity. Methods of assessing the success of ocular cell/stem celltransplantation (e.g., extent of cellular regeneration, visual acuity)are known in the art, any of which may be used in accordance with theinvention. Examples of methods for assessing success of a ocularcell/stem cell transplantation include, without limitation, slit lampimaging, Heidelberg retina tomography (HRT), optical coherencetomography (OCT) and 2-photon imaging. Other examples include, withoutlimitation, the use of Rose Bengal(4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein) dye and otherepithelial staining solutions.

The ABCB5(+) stem cells (e.g., ABCB5(+) LSCS) may be autologous to thesubject (obtained from the same subject) or non-autologous such as cellsthat are allogeneic or syngeneic to the subject. Alternatively, theABCB5(+) stem cells (e.g., ABCB5(+) LSCS) may be obtained from a sourcethat is xenogeneic to the subject.

Allogeneic refers to cells that are genetically different althoughbelonging to or obtained from the same species as the subject. Thus, anallogeneic human ABCB5(+) limbal stem cell is a limbal stem cellobtained from a human other than the intended recipient of the limbalstem cells. Syngeneic refers to cells that are genetically identical orclosely related and immunologically compatible to the subject (i.e.,from individuals or tissues that have identical genotypes). Xenogeneicrefers to cells derived from or obtained from an organism of a differentspecies than the subject.

The ABCB5(+) stem cells (e.g., ABCB5(+) LSCS) in accordance with theinvention may be expanded ex-vivo prior to the administering step. Thus,in some instances, ABCB5 expression provides a basis for identifying,isolating, cloning, propagating, and expanding ABCB5(+) stem cells(e.g., ABCB5(+) LSCS) in vitro. The present invention contemplates anysuitable method of employing agents, e.g., isolated peptides, e.g.,antibodies, that bind to ABCB5 to separate ABCB5(+) stem cells fromother cells. The isolated ABCB5(+) stem cells may be maintained in anappropriate culture environment using, for example, a combination ofmedia, supplements and reagents. Optionally, feeder cell populations orconditioned media obtained from feeder cell populations may be used toexpand the ABCB5(+) stem cell populations.

Adhesion, attachment and matrix factors that may be used for stem cellexpansion in accordance with the invention include, without limitation,E-cadherin, collagen, fibronectin, superfibronectin, heparin sulfateproteoglycan, ICAM-I, laminin, osteopontin, proteoglycan, E-selectin,L-selectin, VCAM and vitronectin.

Bioactives and supplements that may be used for stem cell expansion inaccordance with the invention include, without limitation, enzymes(e.g., cathepsin G, Flt-3/Fc), proteins and peptides (e.g., activin A,albumin, angiogenin, angiopoietin, BAX inhibiting peptide, heregulinbeta-1, SMAC/Diablo), vitamins, hormones and various other substances(e.g., L-ascorbic acid, dexamethasone, EGF, EGF-receptor, embryonicfluid (bovine), flt3-ligand, progesterone, retinoic acid, retinylacetate, thrombopoietin and TPO), antibodies, chemokines, cytokines,growth factors and receptors.

Culture reagents that may be used for stem cell expansion in accordancewith the invention include, without limitation, antibiotics (e.g.,cycloheximide, etoposide, gentamicin, mitomycin,penicillin-streptomycin), classical media (e.g., Claycomb Medium,Dulbecco's Modified Eagle Medium, Iscove's Modified Dulbecco's Medium,Minimum Essential Medium), cell freezing medium-DMSO, Claycomb Mediumwithout L-glutamine, Stemline® Medium (Sigma-Aldrich, USA).

As used herein, a subject may be a mammal such as, for example, a human,non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.Human ABCB5(+) stem cells (e.g., ABCB5(+) LSCs) and human subjects areparticularly important embodiments.

Compositions of the present invention may comprise stem cells (e.g.,limbal stem cells), or an isolated preparation of stem cells, the stemcells characterized by the expression of ABCB5 on their cell surface. Acomposition may comprise a preparation enriched with isolated ABCB5(+)stem cells (e.g., ABCB5(+) LSCs), or it may comprise a substantiallypure population of ABCB5(+) stem cells (e.g., ABCB5(+) LSCs).Compositions are meant to encompass ocular grafts, discussed herein.

The compositions, in some embodiments, may comprises additionalbioactives and supplements to promote cell regeneration anddifferentiation. Such bioactives and supplements that may be used inaccordance with the invention are describe above and include, withoutlimitation, various enzymes, proteins and peptides, vitamins,antibodies, chemokines, cytokines, growth factors and receptors. In someembodiments, the compositions may comprise an immunosuppressant and/oran anti-vasculogenesis agent. For example, in some embodiments, acomposition may comprise cyclosporin (e.g., CyA), which may be used toprevent and/or treat graft rejections. In some embodiments, thecompositions may comprise bevacizumab (e.g., AVASTIN®). The use ofanti-vasculogenesis agent may be used, in some instances, to preventblood vessel formation, which often occurs after transplantation and maylead to graft rejection. In some embodiments, an immunosuppressantand/or an anti-vasculogenesis agent is not administered as a componentof a composition, but rather is administered independently prior to orsubsequent to administration of ABCB5(+) stem cells.

In some embodiments, the compositions are formulated for topicaladministration. An example of a composition formulated for topicaladministration is an ocular graft. An ocular graft for transplantationin accordance with the invention refers to a substrate containingACBC5(+) stem cells (e.g., ACBC5(+) LSCs) and optionally other ocularcells and bioactive factors (e.g., cytokines, growth factors) thatpromote ocular cell regeneration, which substrate may be transplanted toor implanted into an eye of a subject to replace damaged or infectedtissue (e.g., to treat an ocular wound). An ocular graft may contain amixed population of cells including ocular cells such as, for example,corneal and/or retinal cells. In some embodiments, an ocular graft fortransplantation is enriched with ABCB5(+) LSCs.

The cornea is the transparent front part of the eye that covers theiris, pupil and anterior chamber. The cornea, with the anterior chamberand lens, refracts light, with the cornea accounting for approximatelytwo-thirds of the eye's total optical power. The cornea of primates hasfive layers: corneal epithelium (multicellular epithelial tissue layer),Bowman's layer (condensed layer of collagen fibers), corneal stroma(middle layer of collagen fibers, e.g., collagen type I fibrils, andkeratocytes), descemet's membrane (thin layer from which cornealepithelium cells are derived, composed of collagen type IV fibrils) andcorneal endothelium (simple squamous or low cuboidal layer ofmitochondria-rich cells). Compositions, including isolated preparationsand ocular grafts, in accordance with the invention may comprise, inaddition to ABCB5(+) stem cells, any one or more of the cell subtypes ofthe five corneal layers. In some embodiments, the compositions do notcontain any one or more of the cell subtypes of the five corneal layers.

The retina is the light-sensitive layer of tissue lining the innersurface of the eye. The retina itself has several layers of neuronsinterconnected by synapses, including photoreceptor cells such as rods,cones and ganglion cells. Compositions, including isolated preparationsand ocular grafts, in accordance with the invention may comprise, inaddition to ABCB5(+) stem cells, any one or more of the neuronal cellsubtypes of the retina, including retinal epithelial cells of the RPE.In some embodiments, the compositions do not contain any one or more ofthe neuronal cell subtypes of the retina.

The cells of a composition intended for use in transplantation (e.g.,ocular graft) may be allogeneic or syngeneic. In some embodiments, thecells are not skin stem cells (e.g., mesenchymal stem cells). Thus, insome embodiments, the cells of a composition of the invention do notcontain (i.e., exclude) ABCB5(+) mesenchymal stem cells.

In some embodiments, the compositions, including ocular grafts, areenriched with ABCB5(+) stem cells. In some embodiments, the oculargrafts are enriched with ABCB5(+) LSCs. In some embodiments, the oculargrafts are enriched with ABCB5(+) RPE stem cells. For example, an oculargraft is considered to be enriched ABCB5(+) LSCs if the ABCB5(+) limbalstem cell is the predominant cell subtype present in the graft. Forexample, an ocular graft is enriched with ABCB5(+) LSCs if the LSCsoutnumber the other cell subtypes in the graft. In some embodiments, atleast 50% of the cells of the graft are ABCB5(+) stem cells or ABCB5(+)limbal stem cells. For example, in some embodiments, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99% or100% of the cells of the ocular graft are ABCB5(+) stem cells orABCB5(+) limbal stem cells. In some embodiments, less than 15%, lessthan 10%, less than 5% or less than 1% of the cells of an ocular graftare ABCB5(−) cells.

The compositions of the invention may comprise a substrate such as, forexample, a biocompatible material that promotes wound healing, includingbiodegradable scaffolds such as, for example, fibrin gel. Fibrin gelsare typically prepared from fibrogen and thrombin, key proteins involvedin blood clotting. Other examples of substrates that may be used inaccordance with the invention include, without limitation, amnioticmembrane, aminoglycan scaffolds, and adhesives. ABCB5(+) stem cells maybe added to the substrate to form, for example, ocular grafts fortransplantation.

Compositions of the invention may be transplanted to, for example, thesurface of the cornea or the retina. Thus, in some embodiments, thecompositions are administered topically. In instances where a stem cellgraft is transplanted to the eye, the graft may be sutured in place. Inother embodiments, the stem cell compositions are injected. In someembodiments, the compositions are injected intravenously,intraarterially or intravascularly. Other routes of administration arecontemplated. It should be understood that the compositions and/orABCB5(+) stem cells of the invention may be administered with or withouta carrier. Thus, in some embodiments, a substantially pure population ofisolated ABCB5(+) stem cells may be administered to a subject to, forexample, treat an ocular condition.

ABCB5 expression may be used to select ocular cell preparations (e.g.,grafts) for transplantation, thereby permitting the selection of ocularcell preparations enriched with ABCB5(+) stem cells. Such methods inaccordance with the invention include identifying the number of ABCB5(+)stem cells (e.g., ABCB5(+) limbal stem cells) in the ocular cellpreparations, comparing the number of ABCB5(+) stem cells to the totalcell population of the cell preparations, and based on the comparison,selecting the ocular cell preparations for transplantation. The numberof ABCB5(+) stem cells in the ocular cell preparations may be identifiedusing any one or more known molecules that selectively bind to ABCB5.For example, in some embodiments, ABCB5(+) stem cells may be identifiedby contacting the cells with an antibody or other binding molecule thatselectively binds to ABCB5. Viable dyes (e.g., rhodamine or other stemcell marker dyes) may also be used to identify ABCB5(+) stem cells.ABCB5(+) stem cells also can be isolated based on the presence orabsence of other specific markers of interest. For example, agents canbe used to recognize stem cell-specific markers, for instance labeledantibodies that recognize and bind to cell-surface markers or antigenson stem cells can be used to separate and isolate ABCB5(+) stem cellsusing fluorescent activated cell sorting (FACS), panning methods,magnetic particle selection, particle sorter selection and other methodsknown to persons skilled in the art, including density separation.Typically, ocular cell preparations are selected for transplantation ifthey are enriched with ABCB5(+) stem cells (e.g., ABCB5(+) limbal stemcells). Such ABCB5(+) enriched cell preparations increase the success oftransplantation. In some embodiments, ocular cell preparations (e.g.,grafts) may be selected for transplantation if at least 0.03% of thetotal cell population is ABCB5(+). In some embodiments, ocular cellpreparations are selected for transplantation if at least 0.04%, atleast 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least0.09%, at least 0.10%, at least 0.15%, at least 0.20%, at least 0.30%,at least 0.40%, at least 0.50%, at least 0.60%, at least 0.70%, at least0.80%, at least 0.90%, at least 1.0%, at least 2.0%, at least 3.0%, atleast 4.0%, at least 5.0%, at least 10.0%, at least 20.0%, at least30.0%, at least 40.0%, at least 50.0%, at least 60.0%, at least 70.0%,at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, atleast 99.9% or 100% of the total cell population is ABCB5(+).

The ABCB5(+) stem cells of the invention may also be used toprepare/produce artificial grafts such as, for example, artificialcorneal grafts. Such grafts may be made from acellular collagen or otheracellular biocompatible material. In some embodiments, isolate ABCB5(+)stem cells are seeded onto an acellular matrix to produce an artificialgraft such as, for example, an artificial cornea.

Compositions for topical administration such as, for example, an oculargraft may be administered by any means known in the art such as, forexample, those described by Rama, J. et al [5].

An example of a method of the invention follows. ABCB5(+) stem cells areobtained and cultured on fibrin gel (e.g., using lethally irradiatedfeeder cells, e.g., 3T3-J2 cells). A 360° limbal peritomy is performedand the fibrovascular corneal pannus carefully removed. Thefibrin-cultured ABCB5(+) epithelial sheet is placed on the preparedcorneal wound bed spanning the limbus (e.g., about 2-3 mm to reducecompetition with conjunctival ingrowth). The conjunctiva is then suturedover the peripheral fibrin sheet with sutures (e.g., 8.0 vicryl sutures)to protect the border of the sheet and help it to adhere on the surface.The eyelids are kept closed (e.g., with STERI-STRIP™ (3M™ NEXCARE™)) andpatched for one week.

The invention also contemplates using the isolated ABCB5(+) stem cells(e.g., ABCB5(+) limbal stem cells or ABCB5(+) corneal stem cells) toproduce totipotent, multipotent or pluripotent stem cells (e.g., inducedpluripotent stem cells (iPSCs)), from which other cells, tissues and/orwhole animals can develop. Thus, methods for directly reprogramming, orinducing, isolated ABCB5(+) stem cells to become totipotent, multipotentor pluripotent stem cells are provided in some aspects of the invention.The term “reprogramming,” as used herein, refers to a process thatreverses the developmental potential of a cell or population of cells(e.g., an isolated ABCB5(+) stem cell). Thus, reprogramming refers to aprocess of driving a cell to a state with higher developmentalpotential, i.e., backwards to a less differentiated state. The cell tobe reprogrammed can be either partially or terminally differentiatedprior to reprogramming. In some embodiments, reprogramming encompasses acomplete or partial reversion of the differentiation state, i.e., anincrease in the developmental potential of a cell, to that of a cellhaving a totipotent, multipotent or pluripotent state. In someembodiments, reprogramming encompasses driving an isolated ABCB5(+) stemcell to a totipotent, multipotent or pluripotent state, such that thecell has the developmental potential of an embryonic stem cell, i.e., anembryonic stem cell phenotype. Reprogramming also encompasses partialreversion of the differentiation state of a cell to a state that rendersthe cell more susceptible to complete reprogramming to a totipotent,multipotent or pluripotent state when subjected to additionalmanipulations.

Totipotent, multipotent or pluripotent stem cells may be generated fromABCB5(+) stem cells (referred to herein as “reprogrammed ABCB5(+)cells”) using several reprogramming factors. The resultant cells, whichhave a greater developmental potential than the isolated ABCB5(+) stemcells, may then become the source of stem cells for furthermanipulations. A “reprogramming factor” as used herein, refers to adevelopmental potential altering factor, the expression of whichcontributes to the reprogramming of a cell, e.g., an isolated ABCB5(+)stem cell, to a less differentiated or undifferentiated state, e.g., toa cell of a pluripotent state or partially pluripotent state.Reprogramming factors include OCT4, SOX2, KLF 4 and c-MYC (otherwiseknown as the “Yamanaka factors” [32], incorporated herein by referencein its entirety). Other reprogramming factors include, withoutlimitation, SOX 1, SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 5,NR5A2, LIN28, 1-MYC, n-MYC, REM2, TBX3, TERT and LIN28. Any combinationof two or more of the foregoing transcription factors may be used toreprogram isolated ABCB5(+) stem cells. Methods of reprogramming cellsto a totipotent, multipotent or pluripotent state are described byStadtfeld and Hochedlinger [33], incorporated herein by reference in itsentirety.

Reprogrammed ABCB5(+)cells may be used, in some embodiments of theinvention, for basic and/or clinical applications, including diseasemodeling, drug toxicity screening/drug discovery, gene therapy and cellreplacement therapy.

For example, reprogrammed ABCB5(+)cells may be used to treat a varietyof conditions (e.g., genetic conditions) including, without limitation,sickle cell anemia, Parkinson's disease, hemophilia A, heart diseasesuch as ischemic heart disease, Alzheimer's disease, spinal cord injury,stroke, burns, diabetes, osteoarthritis and rheumatoid arthritis.

In some embodiments, the reprogrammed ABCB5(+) cells may be used inorgan transplantations to provide cell types that are geneticallymatched with a patient.

Other basic and clinical uses of the reprogrammed ABCB5(+) stem cellsare contemplated.

Methods for producing differentiated cells from reprogrammed ABCB5(+)cells are also provided herein. The methods may comprise expressing inthe reprogrammed ABCB5(+) cells any one or more differentiation factorsnecessary to promote differentiation into a more mature, differentiatedcell type such as, for example, a blood cell, platelet, stromal cell,bone cell, muscle cell, skin cell, fat cell or neural cell. As usedherein, the term “differentiation factor” refers to a developmentalpotential altering factor such as a protein, or small molecule thatinduces a cell to differentiate to a desired cell-type, e.g., adifferentiation factor reduces the developmental potential of a cell.Differentiation to a specific cell type may involve simultaneous and/orsuccessive expression of more than one differentiation factor. Themethods may further comprise growing the reprogrammed ABCB5(+) cellsunder conditions for promoting differentiation to form a differentiatedcell.

Thus, reprogrammed ABCB5(+) cells can be generated from isolatedABCB5(+) stem cells of the invention (e.g., isolated ABCB5(+) limbalstem cells or isolated ABCB5(+) RPE stem cells), and the reprogrammedABCB5(+) cells can be differentiated into one or more desired celltypes. A “stem cell” as used herein is an undifferentiated or partiallydifferentiated cell that has the ability to self-renew and has thedevelopmental potential to differentiate into multiple cell types. A“pluripotent cell” is a cell with the developmental potential, underdifferent conditions, to differentiate to cell types characteristic ofall three germ cell layers, i.e., endoderm (e.g., gut tissue), mesoderm(including blood, muscle, and vessels), and ectoderm (such as skin andnerve). A “multipotent” cell is a cell that has the developmentalpotential to differentiate into cells of one or more germ layers, butnot all three. These cells include, for instance, adult stem cells, suchas for example, hematopoietic stem cells and neural stem cells. A“totipotent” cell is a cell that has the developmental potential todifferentiate into all the differentiated cells in an organism,including extraembryonic tissues. Stem cells may have a propensity for adifferentiated phenotype; however, these cells can be induced to reverseand re-express the stem cell phenotype. This process is referred to as“dedifferentiation” or “reprogramming.”

The isolated ABCB5(+) stem cells, reprogrammed ABCB5(+) cells anddifferentiated cells of the invention can be manipulated under standardconditions for these cell types. The treatment of the cells may beperformed in vitro, ex vivo or in vivo. For instance, the cells may bepresent in the body or in a culture medium. The manipulations may beperformed under high or low-oxygen conditions.

A “culture medium” contains nutrients that maintain cell viability andsupport proliferation. A typical culture medium includes: salts,buffers, amino acids, glucose or other sugar(s), antibiotics, serum orserum replacement, and/or other components such as peptide growthfactors. Cell culture media for use in deriving and maintainingtotipotent, multipotent and pluripotent cells are known in the art.Culture medium may also include cell specific growth factors, such asangiogenin, bone morphogenic protein-1, bone morphogenic protein-2, bonemorphogenic protein-3, bone morphogenic protein-4, bone morphogenicprotein-5, bone morphogenic protein-6, bone morphogenic protein-7, bonemorphogenic protein-8, bone morphogenic protein-9, bone morphogenicprotein-10, bone morphogenic protein-11, bone morphogenic protein-12,bone morphogenic protein-13, bone morphogenic protein-14, bonemorphogenic protein-15, bone morphogenic protein receptor IA, bonemorphogenic protein receptor IB, brain derived neurotrophic factor,ciliary neutrophic factor, ciliary neutrophic factor receptor-alpha,cytokine-induced neutrophil chemotactic factor 1, cytokine-inducedneutrophil, chemotactic factor 2-alpha, cytokine-induced neutrophilchemotactic factor 2-beta, beta-endothelial cell growth factor,endothelia 1, epidermal growth factor, epithelial-derived neutrophilattractant, fibroblast growth factor 4, fibroblast growth factor 5,fibroblast growth factor 6 fibroblast growth factor 7, fibroblast growthfactor 8, fibroblast growth factor b, fibroblast growth factor c,fibroblast growth factor 9, fibroblast growth factor 10, fibroblastgrowth factor acidic, fibroblast growth factor basic, glial cellline-derived neutrophil factor receptor-alpha-1, glial cell line-derivedneutrophil factor receptor-alpha-2, growth related protein, growthrelated protein-alpha, growth related protein-beta, growth relatedprotein-gamma, heparin binding epidermal growth factor, hepatocytegrowth factor, hepatocyte growth factor receptor, insulin-like growthfactor I, insulin-like growth factor receptor, insulin-like growthfactor II, insulin-like growth factor binding protein, keratinocytegrowth factor, leukemia inhibitory factor, leukemia inhibitory factorreceptor-alpha, nerve growth factor, nerve growth factor receptor,neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growthfactor 2, platelet-derived endothelial cell growth factor, plateletderived growth factor, platelet derived growth factor A chain, plateletderived growth factor AA, platelet derived growth factor AB, plateletderived growth factor B chain, platelet derived growth factor BB,platelet derived growth factor receptor-alpha, platelet derived growthfactor receptor-beta, pre-B cell growth stimulating factor, stem cellfactor, stem cell factor receptor, transforming growth factor-alpha,transforming growth factor-beta, transforming growth factor-beta-1,transforming growth factor-beta-1-2, transforming growth factor-beta-2,transforming growth factor-beta-3, transforming growth factor-beta-5,latent transforming growth factor-beta-1, transforming growthfactor-beta-binding protein I, transforming growth factor-beta-bindingprotein II, transforming growth factor-beta-binding protein III, tumornecrosis factor receptor type I, tumor necrosis factor receptor type II,urokinase-type plasminogen activator receptor, vascular endothelialgrowth factor, and chimeric proteins and biologically or immunologicallyactive fragments thereof.

The differentiation state of the cell can be assessed using any methodsknown in the art for making such assessments. For instance, thedifferentiation state of a cell treated according to the methodsdescribed herein may be compared with an untreated cell or cells treatedwith DNA using viral vectors to deliver DNA resulting in the expressionof the same reprogramming or differentiation factors.

The following examples are provided to illustrate specific instances ofthe practice of the present invention and are not intended to limit thescope of the invention. As will be apparent to one of ordinary skill inthe art, the present invention will find application in a variety ofcompositions and methods.

Examples

C57BL/6J, NOD.Cg-Prkdcscid Il2rgtmIWjl/SzJ (NSG),B6;SJL-Tg(ACTFLPe)9205Dym/J, and B6.FVB-Tg(EIIa-cre)C5379Lmgd/J micewere purchased from Jackson Laboratory (Bar Harbor, Me.). Abcb5 knockout(KO) mice were generated as described below. All animals were maintainedin accordance with the Institutional Guidelines of Boston Children'sHospital and the Schepens Eye Research Institute, Harvard MedicalSchool. Four to twelve weeks-old mice were used for the followingexperiments.

Example 1 ABCB5 is a Molecular Marker of Limbal Stem Cells (LSCs)

To investigate whether ABCB5 is a marker of slow cycling,label-retaining limbal stem cells in the mammalian eye, in vivoBrdU-based ‘pulse and chase’ experiments [2] were performed, in whichAbcb5 wild type (WT) mice were subjected over a 9-day period to dailysystemic BrdU administration in order to label slow-cycling cells(pulse), followed by an 8-week BrdU-free period (chase) prior toevaluation for limbal stem cell label retention (FIG. 5A). Flowcytometric analysis of dissociated murine corneal and limbal epithelialcells revealed BrdU label-retaining cells to be detectable in thelimbus, but not in the central cornea (FIG. 1B and FIG. 5B). BrdUimmunohistochemical staining of full thickness murine corneas confirmedlabel-retaining limbal stem cells (LSCs), consistent with previousfindings [2], to be located in the basal layer of murine limbalepithelium (FIG. 1C). Moreover, label-retaining LSCs expressed ABCB5(FIG. 1C). Flow cytometric quantification confirmed ABCB5(+) cells to bepredominantly BrdU-positive (90.5±0.5%, mean±s.e.m.), withABCB5/BrdU-double positive cells comprising 1.8% of all limbalepithelial cells 4 (FIG. 1D).

Similar to findings in mice, human ABCB5(+) cells were also located inthe basal layer of the limbal epithelium (FIG. 1E and FIG. 6), andimmunohistochemical analysis revealed that ABCB5(+) cells co-expressedthe limbal stem cell marker ΔNp63α (FIGS. 1F and 1G), absent expressionof the corneal differentiation marker KRT12 (FIG. 1H). Flow cytometryalso revealed that ABCB5(+) cells, but not ABCB5(−) cells, expressedsignificant levels of ΔNp63α (28.9±5.7% and 0.1±0.1%, respectively,P=0.0364) (FIG. 1G) and showed that essentially all ΔNp63α(+) LSCsexpressed ABCB5 (ΔNp63α(+) LSCs: 95.3±4.8%, ΔNp63α(−) cells: 3.6±2.1%,P=0.0032). Further, human limbal stem cell deficiency (LSCD) patientsexhibited significantly reduced ABCB5(+) frequencies compared to healthydonors (2.8±1.6% and 20.0±2.6%, respectively, P<0.0001) (FIG. 1I, FIGS.7 and 8, Table 1).

TABLE 1 LSCD patient information Cause of Other Previous Patient GenderAge LSCD Pathology surgery Procedure 1* Male 46 Chemical burn - GlaucomaNone KLAL + PKP OD suspect OD 2** Female 31 Autoimmune Multiple 2 × PKPsKLAU corneal melt; graft failure Cataract PUK with OD surgery partialLSCD Retinal vasculitis OD *Donor 1: cadaveric donor **Donor 2:autologous transplant from contralateral eye Abbreviations: PKPPenetrating keratoplasty KLAL Kerato-limbal allograft (limbal tissue washarvested from donor eye) KLAU Kerato-limbal autograft (part of limbaltissue was resected from uninjured contralateral eye) PUK Peripheralulcerative keratitis OD Right eye

The expression of ABCB5 on label-retaining limbal stem cells in Abcb5 WTmice and ΔNp63α(+) LSCs in healthy humans, and the concurrent finding ofreduced ABCB5(+) cell frequency in clinical LSCD patients, showed thatABCB5 marks LSCs.

Example 2 ABCB5 Regulates Corneal Development and Regeneration

To investigate a potential functional role of ABCB5(+) LSCs in cornealdevelopment and regeneration, Abcb5 KO mice carrying a deletion of exon10 of the murine Abcb5 gene (GenBank JQ655148) were generated. Exon 10the murine Abcb5 gene encodes a functionally critical extracellulardomain of the molecule homologous to extracellular loop-associated aminoacid residues 493-508 of human ABCB5 (GenBank NM_(—)178559).

A conditional knockout targeting construct was first generated byrecombineering (i.e., recombination-mediated genetic engineering) [25].Briefly, a neomycin resistance cassette flanked by two loxP sites (basedon plasmid pL-452) was inserted into the BAC clone RP23-161L22 458 basepairs upstream of exon 10 of the murine Abcb5 gene (GenBank accessionnumber JQ655148) (FIGS. 2A and 2B). The targeted region of the BAC clonewas retrieved by gap repair into the pL-253 plasmid. The retrievedplasmid contained 6006 base pairs upstream of exon 10 (not including theinserted neo cassette) and 6384 base pairs downstream of exon 10. Theneomycin resistance cassette was excised by arabinose induction of Crerecombinase to leave a single loxP site upstream of exon 10. A neomycinresistance cassette flanked by two FRT sites and one loxP site (based onplasmid pL-451) was inserted 460 base pairs downstream of exon 10 tocomplete the targeting construct. The targeting plasmid was verified byDNA sequencing and restriction mapping. The linearized plasmid wastransfected into TC1 (129S6/SvEvTac derived) embryonic stem (ES) cellsand selected in G418 (Sigma-Aldrich, MO) and Fialuridine (MoravekBiochemicals, CA). Resistant colonies were expanded and screened bylong-range PCR to identify targeted clones [22]. The left arm wasamplified with 5′-GTTGAGGGGAGCAGCCAGAGCAAGGTGAGAAAGGTG-3′(SEQ ID NO:1)and 5′-TTAAGGGTTATTGAATATGATCGGAATTGGGCTGCAGGAATT-3′(SEQ ID NO:2)primers yielding a 6250 base pair PCR product (FIG. 2B). The right armwas amplified with 5′-TGGGGCAGGACAGCAAGGGGGAGGAT-3′ (SEQ ID NO:3) and5′-CTGGTCCCTCTCCTGTGATCTACACAGGCC-3′ (SEQ ID NO:4) primers yielding a6384 base pair PCR product (FIG. 2B). Two Abcb5-targeted ES clones wereidentified. These clones were expanded and injected into C57BL/6blastocysts that were then transferred to the uterus of pseudo-pregnantfemales. High-percentage chimeric male mice (Abcb5^(neo-loxP/wt)) werebred into a C57BL/6 background to obtain germ-line transmission.Germ-line transmission of the Abcb5^(neo-loxP) allele was confirmed byPCR analysis of genomic DNA using 5′-GGAAGACAATAGCAGGCATGCTGGG-3′ (SEQID NO:5), 5′-GGCTGGGGCAACTGAAAAGTAGCAT-3′ (SEQ ID NO:6), and5′-TTTCAGCTTCAGTTTATCACAATGTGGGTT-3′ (SEQ ID NO:7) primers designed toamplify the 385 base pair targeted allele and the 284 base pair WTallele. Heterozygous Abcb5^(neo-loxP) mice were then intercrossed withhACTB-FLPe transgenic mice [26] to remove the neomycin resistancecassette. PCR analysis of genomic DNA was performed to confirm removalof the neomycin resistance cassette in the genome of Abcb5^(loxP/wt)mice using 5′-ACTT GGTGCGGTGACTCTGAATTTTGC-3′ (SEQ ID NO:8) and5′-TAGCAACATTTCTGGCATTTTAGGCTG-3′ (SEQ ID NO:9) primers designed toamplify a 494 base pair neomycin resistance cassette-deleted allele anda 390 base pair WT allele. Abrogation of ABCB5 protein expression inAbcb5 KO animals was determined by Western blots of murine tissues (FIG.2C). Abcb5 WT and Abcb5 KO cell lysates were immunoblotted usingmonoclonal ABCB5 antibody 3C2-1D12 [6,27] (5.5 m/ml) or α-Tubulin rabbitpolyclonal antibody (1:5000 dilution) (Abcam, MA). After treatment withHRP-conjugated specific secondary antibodies (1:5000 dilution) (JacksonImmunoResearch, PA), signals were visualized on film by enhancedchemiluminescence.

To determine the outcome of a complete loss of ABCB5 function, exon 10of the murine Abcb5 gene was deleted by breeding Abcb5^(loxP) mice withElla-Cre mice, which express Cre recombinase at the zygote stage [14,15](FIG. 2B). Deletion of the genomic region between the two loxP sites wasconfirmed by PCR analysis of genomic DNA using5′-GGCTGGGGCAACTGAAAAGTAGCAT-3′ (SEQ ID NO:10),5′-GCAAATGTGTACTCTGCGCTTATTTAATG-3′ (SEQ ID NO:11) and5′-TGGTGCAGACTACAGACGTCAGTGG-3′ (SEQ ID NO:12) primers designed toamplify a 322 base pair cre-deleted allele (null) and al 13 base pair WTallele (FIG. 2C). Heterozygous Abcb5^(null/WT) mice with the germlinedeletion of exon 10 were intercrossed to produce homozygousAbcb5^(null/null) mutants (Abcb5 KO mice). Mice were maintained on a129S6/SvEvTac/C57BL/6 mixed genetic background, and littermates wereused as controls for experimental analyses.

Abcb5 KO mice were born alive and appeared indistinguishable from theirWT littermates at birth upon physical examination, with no grossanatomical defects of Abcb5 KO corneas detectable by slit lampexamination (FIG. 2D). However, histological analysis of mutant corneasdemonstrated profound developmental abnormalities characterized byflattening of the corneal epithelium compared to WT controls, withsignificantly reduced epithelial cell numbers in the central cornea, butnot in the limbus, as evidenced by hematoxylin and eosin (H&E) stain,4′,6-diamidino-2-phenylindole (DAPI) staining and flow cytometry(Central cornea: 2688±399 cells and 4427±346 cells, respectively,P=0.0165; limbus: 3015±433 cells and 3629±94 cells, respectively,P=0.2377) (FIG. 2D and FIG. 9). Abcb5 KO corneas also exhibited severeepithelial tight junction defects as determined by LC biotin staining(FIG. 2E), and mutant mice showed significantly decreased limbal andcorneal PAX6 and corneal KRT12 expression as compared to WT mice (limbalPAX6: 0.3±0.3% and 18.0±4.6%, respectively, P=0.0181; corneal PAX6:8.3±4.6% and 42.0±7.6%, respectively, P=0.0192; corneal KRT12: 6.5±6.5%and 47.7±8.2%, respectively, P=0.0382) (FIG. 2E, FIG. 10), demonstratinga novel essential role of ABCB5 in normal corneal development.

Example 3 ABCB5 Regulates Limbal Stem Cell Quiescence

To determine whether corneal regeneration is dependent on intact ABCB5function, Abcb5 KO and WT mice were subjected to central cornealepithelial debridement injury followed by evaluation for cornealregeneration (FIGS. 11A-11D). After anesthesia with intraperitonealinjection of Ketamine (120 mg/kg body weight, Hospira, IL) and Xylazine(10 mg/kg body weight, Burns Veterinary Supply, NY), followed by topicalapplication of one drop of 0.5% Proparacaine eye drops (Akorn, IL) intoeach eye, a 2 mm diameter epithelial wound was created by demarcating anarea of the central cornea with a 2 mm trephine and removing theepithelium within the circle with a small scalpel, leaving the basementmembrane intact. In each animal, the procedure was performed on theright eye. Ak-Spore Ophthalmic Ointment (Bacitracin Zinc, NeomycinSulfate and Polymyxin B Sulfate, Akorn, IL) was applied to both eyesimmediately after wounding and then twice per day for the next 48 hoursto prevent corneal infection and dryness. Analgesia was provided bysubcutaneous injections of Buprenex (Reckitt Benckiser Pharmaceuticals,Berkshire, 30 UK) every 12 hours for 48 hours postoperatively at thedose of 1 mg/kg. The wound healing was monitored as described previously[29]. Animals were euthanized 48 hours post-operatively and theintegrity of corneal epithelial tight junctions was assessed using theLC-Biotin staining method performed as described [31]. Briefly,LC-Biotin staining solution prepared by dissolving 1 mg/mlEZ-Link-Sulfo-NHS-LC-Biotin (Pierce, IL) in HBSS (Hank's Balanced SaltSolution, Lonza, MD) plus 2 mM MgCl₂, and 1 mM CaCl₂ was applied towounded and non-wounded eyes for 15 minutes at the time of euthanasia.Eyes were rinsed with PBS (Lonza, MD), enucleated and placed inTissue-Teck OCT (Sakura Finetek, CA) for frozen sectioning.

While no significant differences were observed in the rate of woundclosure between Abcb5 WT and Abcb5 KO mice (FIGS. 11C and 11D),histological analysis revealed severely abnormal corneal restoration inAbcb5 KO mice, as compared to Abcb5 WT mice, characterized by highlyirregular appearance of the epithelium with reduced number of epithelialcells (403.3±29.7 and 737.2±28.2, respectively, P<0.0001) (FIG. 2F andFIG. 12), significantly increased cellular proliferation as demonstratedby enhanced Ki67 expression (limbus: 54.0±5.0% and 0.3±0.2%,respectively, P<0.0001; cornea: 41.2±12.8% and 1.0±0.5%, respectively,P=0.0257) (FIG. 2G), and significantly enhanced rates of apoptosis asdemonstrated by TUNEL staining (limbus: 41.2±12.8% and 1.0±0.5%,respectively, P=0.001; cornea: 49.0±1.0% and 0.4±0.3%, respectively,P<0.0001) (FIG. 2H and FIG. 13).

Pulse-chase BrdU-labeling (FIGS. 5A and 5B) and flow cytometric analysisof dissociated murine limbal epithelial cells revealed that after anearly, 1-week chase period, no significant difference existed betweenthe numbers of BrdU-labeled epithelial cells in Abcb5 KO-derived andAbcb5 WT-derived specimens, indicative of equal BrdU uptake by Abcb5 KOand Abcb5 WT limbal cells (1.9±0.7% and 1.5±0.4%, respectively,P=0.6971). By contrast, after an 8-week chase period, label-retainingLSC frequency was markedly and significantly reduced (by 89%) in Abcb5KO mice, compared to Abcb5 WT controls (frequency: 0.1±0.1% and0.9±0.3%, respectively P=0.0152) (FIGS. 3A and 3B), demonstrating thatabrogation of ABCB5 function induces cellular proliferation of normallyquiescent LSCs. Consistent with this result, Ki67 expression, indicativeof cellular proliferation, was significantly enhanced in Abcb5 KOcorneas, as compared to Abcb5 WT control corneas (limbus: 24.0±5.0% and1.5±1.5%, respectively, P<0.0001; cornea: 53.0±16.0% vs. 11.0±2.1%,P=0.0297) (FIG. 3C). Moreover, in line with demonstrated increasedproliferation, real-time quantitative PCR (qPCR) analysis of RNAexpression revealed significant down-regulation in Abcb5 KO cornealepithelial cells of the p53 family (p53 and p63) and the Cip/Kip family(p21 and p27) of cell cycle regulators, which control the G0/G1 cellcycle checkpoint and cellular quiescence, as compared WT controls(41.6±16.4% of WT p53, P=0.0377; 31.2±13.8% of WT p63, P=0.0155;37.2±13.8% of WT p21, P=0.0197; 36.8±7.0% of WT p27, P=0.0029) (FIG.3D). Thus, ABCB5 is required for the maintenance of slow-cycling LSCs.Because withdrawal from the cell cycle is a prerequisite for LSCmaintenance, and hence normal differentiation, these results provide anexplanation for the observed corneal differentiation defect in Abcb5 KOmice (FIG. 3E).

Example 4 Regenerative Role of ABCB5(+) Limbal Stem Cells in Treatmentof Limbal Stem Cell Deficiency

To investigate whether ABCB5 represents a molecular marker forprospective enrichment of limbal stem cells within grafts to improvetransplantation outcomes, the cornea-regenerative potential oftransplanted limbal epithelial cells was examined. (FIGS. 14A and 14Band FIGS. 15A-15C). Murine donor limbal epithelial cells weretransplanted onto the eyes of syngeneic C57BL/6J recipient mice with aninduced limbal stem cell deficiency (LSCD). Human donor limbalepithelial cells were transplanted onto the eyes of immunodeficientNOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl/SzJ) (NSG) mice with an induced limbalstem cell deficiency. Four types of donor transplants were performed:(i) ABCB5(+) limbal epithelial cells, (ii) ABCB5(−) limbal epithelialcells, (iii) unsegregated limbal epithelial cells, and (iv) graftscontaining no cells (fibrin gel carrier only) (500 cells in fibrin gelvehicle/graft, 1 unilateral eye graft/mouse, n=5 mice/treatment group)(Table 2). Three days

TABLE 2 Number and viability of donor cells used for transplantationABCB5(−) cells ABCB5(+) cells (ABCB5-negative Unsegregated(ABCB5-positive enriched) cells enriched) 500 cells/ 500 cells/ 500cells/ graft/mouse graft/mouse graft/mouse ABCB5 ABCB5 ABCB5 Nega- DonorPositive Negative Positive Negative Positive tive Mouse limbus % of0.367 99 51 43 0 99 cells/graft viable 69 64 100 40 0 63 cells/graft (%)viable 1 319 255 86 0 312 cells/graft (number) Human limbus % of 0.03 9959 40 0 100 cells/graft viable 93 22 99 40 0 90 cells/graft (%) viable 1109 292 80 0 450 cells/graft (number)prior to transplantation, murine and human donor cells were seeded ontoa fibrin carrier, which was prepared by dissolving fibrinogen andthrombin stock solutions (TISSUCOL-Kit Immuno, Baxter, Germany) in 1.1%NaCl and 1 mM CaCl2 to a final concentration of 10 mg/ml fibrinogen and3 IU/ml thrombin as described [30]. On the day of transplantation, totalLSCD was induced in anesthetized recipient mice by removing the cornealand limbal epithelium with an Algerbrush II corneal rust ring removerwith a 0.5-mm burr (AMBLER Surgical, PA) [16]. Following induction ofLSCD, recipient mice received fibrin gel carrier-based transplants thatwere secured through four sutures. Eyelids were sutured with 8-0 nylonsutures to keep the eyes closed. Ak-Spore Ophthalmic Ointment(Bacitracin Zinc, Neomycin Sulfate and Polymyxin B Sulfate, Akorn, IL)was applied on both eyes immediately after wounding and then twice perday for the next 48 hours to prevent corneal infection and dryness.Analgesia was provided by subcutaneous injections of 5-10 mg/kg Metacam(Boehringer Ingelheim Pharmaceuticals, CT), given preoperatively and bysubcutaneous injections of 0.05-0.1 mg/kg of Buprenex (Reckitt BenckiserPharmaceuticals, Berkshire, UK) every 12 hours for 24 hourspostoperatively. In addition, after surgical recovery, mice were alsotreated with anti-inflammatory Inflanefran Forte eye drops (Allergan,MA) for the first 5 days, and then with 1% Avastin (Bevacizumab,Genentech, CA) eye drops daily for 5 days. Slit lamp examination wasperformed weekly until euthanasia. Eyes were enucleated postmortem andfixed in 10% buffered formalin for methacrylate embedding (Technovit,Heraeus Kulzer, Germany) or snap-frozen in Tissue-Teck OCT (SakuraFinetek, CA).

Recipients of syngeneic murine Abcb5(−) limbal cell grafts orvehicle-only negative controls displayed opaque corneas, epithelialconjunctivalization with infiltrating goblet cells, and absence ofdifferentiated KRT12(+) cells (0%, respectively) when analyzed 5-weekspost transplantation, consistent with persistent LSCD (FIG. 4A, FIG.16). Recipients of syngeneic grafts containing unsegregated limbal cellsdisplayed partial corneal restoration with detectable differentiatedKRT12(+) cells in the central cornea (17% of cells, significantlyenhanced compared to Abcb5(−) or vehicle-only treatment regimens,P<0.01), but exhibited persistence of LSCD-characteristic goblet cellsand epithelial conjunctivalization (FIG. 4A, FIG. 16). By contrast,syngeneic ABCB5(+) limbal cell grafts resulted in the development ofclear corneas with normal histology in recipient mice, gave rise tohigher numbers of differentiated KRT12(+) corneal epithelial cells (47%of cells, significantly increased compared unsegregated or ABCB5(−)limbal cell treatment regimens or compared vehicle-only controls,P<0.001) and prevented goblet cell formation or epithelialconjunctivalization (FIG. 4A, FIG. 16).

Immuno-compromised NSG recipients of freshly isolated human ABCB5(−)limbal cell grafts or vehicle-only negative controls also displayedepithelial conjunctivalization and absence of differentiated KRT12(+)cells (0%, respectively) 5-weeks post transplantation, consistent withpersistent LSCD (FIG. 4B, FIGS. 17 and 18). Immuno-compromised NSGrecipients of freshly isolated human unsegregated limbal cell grafts,similar to findings in murine unsegregated limbal cell transplantationexperiments, displayed partial corneal restoration with detectability ofdifferentiated KRT12(+) cells in the central cornea (12% of cells,significantly enhanced vs. ABCB5(−) or vehicle-only treatment regimens,P<0.01), but exhibited persistence of LSCD characteristic epithelialconjunctivalization (FIG. 4B, FIG. 17). Strikingly, only freshlyisolated human ABCB5(+) limbal cell grafts resulted in the developmentof clear corneas with normal histology in recipient NSG mice withpresence of a stratified epithelial layer containing high numbers ofKRT12+ cells (31% of cells, significantly increased compared tovehicle-only or compared to ABCB5(−) or unsegregated limbal celltreatment regimens, P<0.001) and absence of LSCD characteristicepithelial conjunctivalization (FIG. 4B, FIG. 17).

In order to confirm that human donor cells had caused cornealrestoration in this xenotransplantation model, regenerated cornealtissue was assayed by RT-PCR for expression of human-specific β2microglobulin (β2M), an identifier of all cells of human origin, and forexpression of human-specific PAX6 and KRT12 as markers of cornealdifferentiation. Only corneal epithelium of recipients grafted withhuman ABCB5(+) or unsegregated human limbal cells containedhuman-specific β2M, PAX6 and KRT12 transcripts, whereasvehicle-only-grafted control eyes that did not exhibit cornealrestoration did not, confirming human specificity of the RT-PCR assaysystem (FIG. 4B). Moreover, despite similar viability in ABCB5(−)compared to unsegregated or ABCB5(+) cell grafts (Table 2, FIGS.15A-15C), ABCB5(−) cell-grafted eyes were deficient in human-specificβ2M, PAX6 or KRT12 transcript expression (FIG. 4B), indicating thatlong-term engraftment capacity is exclusively contained within the humanABCB5(+) limbal cell population.

The Examples provided herein demonstrate that ABCB5(+) cell frequency isreduced in limbal stem cell deficiency (LSCD), that ABCB5-positivitypreferentially characterizes slow-cycling and ΔNp63α-positivepopulations enriched for limbal stem cells (LSCs), and thatprospectively isolated ABCB5(+) limbal cells are exclusively capable ofreversing LSCD, indicating that ABCB5-positivity defines LSCs. Thesefindings are further supported by data demonstrating that ABCB5 loss offunction in Abcb5 gene knockout (KO) mice causes LSCD and impairsLSC-dependent corneal development and regeneration, through abrogationof LSC self-renewal capacity. These results have several importantimplications.

First, successful enrichment of human LSCs has the potential todecisively advance the field of LSCD therapy, because long-term clinicalsuccess has been shown to depend on limbal stem cell frequency withingrafts[5] and because, prior to the present invention, no marker forprospective limbal stem cell enrichment has been available. Indeed,these Examples show that prospective limbal stem cell enrichment withingrafts can significantly enhance LSCD therapeutic success. ABCB5expression on the limbal stem cell surface permits monoclonalantibody-based cell sorting strategies and significant limbal stem cellenrichment as demonstrated herein, unlike intracellularly expressedΔNp63α or alternative candidate limbal stem cell markers [17] that havenot been successfully employed for prospective isolation of LSCs capableof LSCD reversal. This underscores the promise of ABCB5 as a potentialmarker for limbal stem cell isolation also for clinical limbal stem celltransplantation.

Second, the data provided herein reveal a novel in vivo physiologicalrole of ABCB5 in the maintenance of stem cell quiescence. Specifically,abrogation of ABCB5 function in newly created Abcb5 KO mice resulted inloss of slow-cycling LSC with inhibited expression of moleculesregulating G0/G1 cell cycle progression, including the limbal stem cellmarker ΔNp63α. This explains the observed co-expression of ABCB5 withΔNp63α by normally quiescent LSCs, and provides an explanation forinduction of limbal stem cell proliferation and apoptosis associatedwith reduction of differentiated cells observed in Abcb5 KO corneas,because the ability of a cell to withdraw from the cell cycle iscritical for both stem cell pool preservation and normaldifferentiation.

Additional Materials and Methods:

BrdU Pulse and Chase Experiments.

Four-week old Abcb5 KO mice and their Abcb5 WT littermates weresubjected to daily intraperitoneal injections of 50 mg/kgBromodeoxyuridine (BrdU, BD Pharmingen, CA) for 9 consecutive days (FIG.5A). Corneal and limbal epithelial cells isolated from Abcb5 WT andAbcb5 KO mice sacrificed at either one week or eight weeks afterreceiving the last BrdU injection were analyzed by flow cytometry andimmunofluorescence. Limbal and central corneal epithelial cells fromage-matched Abcb5 WT and Abcb5 KO littermates were used as experimentalcontrols. Flow cytometry and immunohistochemistry staining were used todetermine the frequency of BrdU-positive and BrdU-negative cells withinepithelia of the limbus and central cornea.

Human and Murine Corneal Cell Isolation.

Cadaveric human corneoscleral tissues derived from consented donors wereobtained from Heartland Lions Eye Banks (Kansas City, Mo.), BascomPalmer Eye Institute (Miami, Fla.), and Carver College of Medicine (IowaCity, Iowa). After removal of the scleral rim, iris and trabecularmeshwork, the limbus and central cornea were dissected under amicroscope. Limbal and central corneal tissues were subsequentlyincubated with 2.4 units/ml Dispase II (Roche Diagnostics, IN) at 37° C.for 1 hour, followed by incubation with 0.5M EDTA (Invitrogen, CA) at37° C. for ten 5-minute cycles to recover the epithelial cells [22,23].Murine limbal and corneal epithelial cells were obtained from Abcb5 KOand Abcb5 WT mice as follows. Immediately after euthanasia by CO²narcosis and subsequent eye enucleation, limbal and central cornealtissues were removed with micro scissors under a dissecting microscope,placed in low Ca²⁺ Keratinocyte Serum Free Medium (KSFM, Invitrogen, CA)and centrifuged for 5 min at 250 g at 4° C. After removal of thesupernatant, tissue pellets were digested in 0.5% Trypsin solution(Lonza, MD) [24]. For transplantation experiments, ABCB5(+) and ABCB5(−)limbal epithelial cells were isolated by Fluorescence Activated CellSorting (FACS) using ABCB5 monoclonal antibody (mAb) labeling [18].Briefly, either human or murine limbal epithelial cells were labeledwith primary ABCB5 mAb (20 μg/μl) for 30 minutes at 4° C., washed toremove excess antibody, followed by a 30 minute incubation withsecondary anti-mouse FITC conjugated IgG. The ABCB5(+) and ABCB5(−)sorting gates were established on a Modified Digital Vantage cell sorter(Becton Dickinson and MGH Pathology Flow Cytometry Core, SimchesResearch Building, Boston) as displayed in FIGS. 15A-15C. Only viablecells were selected for sorting by excluding all DAPI(+) cells (1 μg/mlDAPI, Sigma-Aldrich, MO, added immediately prior to sorting) asidentified using a 70 MW UV laser for excitation. The purity andviability of ABCB5(+) and ABCB5(−) sorted cells were established inrepresentative post sort analyses in which samples were re-analyzed(FIGS. 15A-15C). ABCB5(+) cell purification resulted in a 255-foldincrease for murine ABCB5(+) limbal cells (0.37% positivity before and51% positivity after sorting, Table 2) and a 292-fold increase for humanABCB5(+) limbal cells (0.03% positivity before and 59% positivity aftersorting, Table 2). ABCB5(−) cell enrichment resulted in complete absenceof ABCB5(+) cells in both mouse and human samples (Table 2).

Flow Cytometric Analysis.

Dual-color flow cytometry was used to determine whether human ABCB5(+)limbal epithelial cells co-expressed ΔNp63α or KRT12 and whether murineABCB5(+) limbal epithelial cells co-expressed PAX6 and KRT12, and wasperformed as described previously [18]. For human and murine ABCB5 andKRT12 co-expression analysis, cells were first incubated with mouseanti-ABCB5 mAb, counterstained with goat anti-mouse FITC IgG, followedby incubation with goat polyclonal anti-KRT12 antibody andcounterstaining with Dylight 649 donkey anti-goat IgG. For human ABCB5and ΔNp63α co-expression and murine ABCB5 and PAX6 co-expressionanalysis, cells were incubated with mouse anti-ABCB5 mAb, counterstainedwith goat anti-mouse FITC IgG, permeabilized in BD Cytofix/CytopermBuffer (BD Biosciences, CA), stained with either ΔNp63α or PAX6 Abs, andcounterstained with goat anti-rabbit Alexa 647 IgG. Washing steps withstaining buffer or BD Perm/Wash Buffer (BD Biosciences, CA) wereperformed between each step. Dual-color flow cytometry was performed byacquisition of fluorescence emission at the Fl1 (FITC) and Fl4 (Alexa647 and/or Dylight 649) spectra on a Becton Dickinson FACScan (BectonDickinson, NJ), as described [18]. Murine ABCB5 and BrdU co-expressionanalysis was performed using the FITC BrdU Flow Kit (BD Biosciences,CA), according to the manufacturer's instructions. Statisticaldifferences between expression levels of the above-listed markers byABCB5(+) and ABCB5(−) cells were determined using the unpaired t test. Atwo-sided P value of P<0.05 was considered significant.

RT-PCR and Quantitative Real Time PCR.

For cell cycle gene expression analyses, total RNA was isolated fromAbcb5 KO and Abcb5 WT corneas using a RT² qPCR Grade RNA isolation kitand then reverse-transcribed using a RT² First Strand Kit for reversetranscriptase-PCR according to the manufacturer's protocol(SABiosciences, CA). Samples were assayed using SYBR Green qPCR MasterMixes (SABiosciences, CA), murine cell cycle arrays (catalog numberPAMM-020Z, SABiosciences, CA) and kinetic PCR (ABI 7700 SequenceDetector; Applied Biosystems, CA), as described [28]. Allquantifications were normalized to the endogenous control genesglyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin, to accountfor variability in the initial concentration and quality of the totalRNA, and efficiency of the reverse transcription reaction. Statisticaldifferences in gene expression levels between Abcb5 KO and Abcb5 WT micewere determined using the one sample t test. A two-sided P value ofP<0.05 was considered significant. For detection of human-specific genetranscripts, total RNA was isolated from transplanted murine eyes andnon-injured murine or human control corneas using the RNAeasy Plusisolation kit (Qiagen, CA) and then transcribed using the High FidelityRT kit (Applied Biosystems, CA). PCR was performed using Taq 2× MasterMix (New England Biolabs, MA) and the following gene-specific primers:human β2-microglobulin (B2M, NM_(—)004048): Forward5′-GTGTCTGGGTTTCATCCATC-3′ (SEQ ID NO:13), Reverse5′-AATGCGGCATCTTCAACCTC-3′ (SEQ ID NO:14); human paired box 6 (PAX6,NM_(—)000280.3): Forward 5′-CAGCGCTCTGCCGCCTAT-3′ (SEQ ID NO:15),Reverse 5′-CATGACCAACACAGATCAAACATCC-3′ (SEQ ID NO:16); human keratin 12(KRT12, NM_(—)000223.3): Forward 5′-GAAGCCGAGGGCGATTACTG-3′ (SEQ IDNO:17), Reverse 5′-GTGCTTGTGATTTGGAGTCTGTCAC-3′ (SEQ ID NO:18); andmurine β-actin (Actb, NM_(—)007393): Forward 5′-TCCTAGCACCATGAAGATC-3′(SEQ ID NO:19), Reverse 5′-AAACGCAGCTCAGTAACAG-3′ (SEQ ID NO:20).

Histopathology and Immunohistochemical Staining.

To recover intact mouse ocular tissue, the whole decapitated mouse headwas fixed in 4% paraformaldehyde (PFA) overnight, then eyes wereenucleated with the lids attached, incubated in 30% sucrose in 1×phosphate buffered saline (PBS) overnight at 4° C., embedded inTissue-Tek OCT compound (Sakura Finetek USA, CA) and snap-frozen.Representative cryostat sections from each tissue block were stainedwith hematoxylin and eosin (H&E). For immunofluorescence staining,cryostat sections (10 μm) were fixed in cold methanol for 10 minutes,blocked in 10% secondary serum+2% bovine serum albumin (BSA) in 1×PBSfor 1 hour, incubated with the primary antibody (or isotype control),followed by the appropriate secondary antibody for 1 hour at roomtemperature. Following several washes, the slides were thencover-slipped in hard-set mounting media with4′,6-diamidino-2-phenylindole (DAPI). BrdU staining was performed usingthe BrdU In-situ Kit (BD Pharmingen, CA) followed by staining withrabbit ABCB5 antibody at 1:250 dilution (NBP1-50547, Novus, CO). TUNELstaining was performed using the In Situ Cell Death Kit (Roche, IN) andDAPI (Invitrogen, MA) was used to stain all nucleated cells. All tissuesections were analyzed using a Nikon Eclipse E800 immunofluorescencemicroscope. Composite corneal photographs were assembled using Photoshop(Adobe) to overlay and match sequential images. Stitching was done byreducing the added photograph to 50% transparency, matching images, andreturning the composite photograph to 0% transparency. The averagenumber of epithelial cells per cornea (FIG. 2D) was determined bycounting the number of DAPI-positive cells within the area defined by a2 mm trephine in a composite photograph of a complete corneal section.At least three composite corneal sections were analyzed per mouse, andfive mice were analyzed per group in four replicate experiments. Thepercentages of epithelial cells expressing Ki67 (FIGS. 2I and 3C), TUNEL(FIG. 2I) and KRT12 (FIGS. 4A and 4B) were determined by counting thenumber of positive cells among the total number of DAPI-positive cornealepithelial cells using the techniques described herein. Comparisonsbetween the Abcb5 WT and Abcb5 KO mice were performed using the unpairedt test. The results of transplantation experiments were compared usingOne-way ANOVA followed by Bonferroni post tests. Differences with P<0.05were considered statistically significant.

Antibodies.

The following primary antibodies were used in flow cytometryexperiments: rabbit polyclonal anti-ΔNp63α antibody (cloneH-129, SantaCruz, Ca.), mouse monoclonal anti-ABCB5 antibody (clone 3C2-2D12) [6],goat polyclonal anti-cytokeratin antibody (clone L15, Santa Cruz,Calif.), rabbit polyclonal anti-PAX6 antibody (Covance, CA), rabbitpolyclonal anti-ABCB5 antibody (Novus Biologicals, CO), rabbitpolyclonal IgG isotype control antibody (Abcam, MA), mouse IgG1k isotypecontrol antibody (BD Biosciences, CA), and goat IgG isotype controlantibody (Santa Cruz, Calif.). The secondary antibodies were goatanti-mouse FITC (Sigma-Aldrich, MO), Alexa 647 goat anti-rabbit IgG(Invitrogen, NY) and Dylight 649 donkey anti-goat (JacksonImmunoResearch, PA). For human histopathology and immunohistochemicalanalyses, the following primary antibodies were used: mouse monoclonalanti-ABCB5 (clone 3C2-1D12) [6] and rabbit antibody against ΔNp63α at1:75 dilution (sc8344, Santa Cruz, Calif.) followed by the appropriatesecondary antibodies obtained from Jackson ImmunoResearch, PA:FITC-donkey anti-rabbit at 1:75 dilution or Alexa Fluor 594-goatanti-mouse at 1:250 dilution. In all cases, isotype-matched antibodiesrabbit IgG (550875, BD Pharmingen, CA) and mouse IgG1kappa isotypecontrol antibody (BD Biosciences, CA) served as negative controls. Forhistopathology and immunohistochemical analyses mouse tissues werestained with the following primary antibodies: rabbit anti-ABCB5antibody at 1:250 dilution (NBP1-50547, Novus, CO), rabbit anti-Pax6 at1:300 dilution (PRB278P, Covance, CA), goat anti-cytokeratin 12 (L15) at1:50 dilution (sc17101, Santa Cruz, Calif.), rabbit anti-cytokeratin 14(AF64) at 1:1000 dilution (PRB-155P, Covance, CA), rabbit anti-Ki67 at1:200 dilution (ab66155, Abcam, MA), followed by the appropriatesecondary antibodies obtained from Jackson ImmunoResearch, PA: donkeyanti-goat Alexa Fluor 488 at 1:250 dilution (705-545-003), donkeyanti-rabbit Alexa Fluor 594 at 1:20 dilution (711-585-152), goatanti-rabbit DyLight 549 at 1:250 dilution (111-504-144), or Cy3-donkeyanti-rabbit at 1:250 dilution (711-165-152). In all cases, isotypedmatched antibodies (rabbit IgG (550875, BD Pharmingen, CA) and goat IgG(sc2028, SantaCruz, CA) served as negative controls.

The concurrently filed Sequence Listing, filed as a text file, isincorporated by reference herein.

REFERENCES

Each of the references listed below is incorporated by reference hereinin its entirety.

-   1. Davanger, M. & Evensen, A. Role of the pericorneal papillary    structure in renewal of corneal epithelium. Nature 229, 560-1    (1971).-   2. Cotsarelis, G., Cheng, S. Z., Dong, G., Sun, T. T. &    Lavker, R. M. Existence of slow cycling limbal epithelial basal    cells that can be preferentially stimulated to proliferate:    implications on epithelial stem cells. Cell 57, 201-9 (1989).-   3. Majo, F., Rochat, A., Nicolas, M., Jaoude, G. A. & Barrandon, Y.    Oligopotent stem cells are distributed throughout the mammalian    ocular surface. Nature 456, 250-4 (2008).-   4. Dua, H. S., Joseph, A., Shanmuganathan, V. A. & Jones, R. E. Stem    cell differentiation and the effects of deficiency. Eye (Lond) 17,    877-85 (2003).-   5. Rama, P. et al. Limbal stem-cell therapy and long-term corneal    regeneration. N Engl J Med 363, 147-55 (2010).-   6. Frank, N. Y. et al. Regulation of progenitor cell fusion by ABCB5    P-glycoprotein, a novel human ATP-binding cassette transporter. J    Biol Chem 278, 47156-65 (2003).-   7. Schatton, T. et al. Identification of cells initiating human    melanomas. Nature 451, 345-9 (2008).-   8. Pellegrini, G. et al. p63 identifies keratinocyte stem cells.    Proc Natl Acad Sci USA 98, 3156-61 (2001).-   9. Li, W. et al. Down-regulation of Pax6 is associated with abnormal    differentiation of corneal epithelial cells in severe ocular surface    diseases. J Pathol 214, 114-22 (2008).-   10. Sun, T. T. & Lavker, R. M. Corneal epithelial stem cells: past,    present, and future. J Investig Dermatol Symp Proc 9, 202-7 (2004).-   11. Liu, C. Y. et al. Characterization and chromosomal localization    of the cornea-specific murine keratin gene Krt1.12. J Biol Chem 269,    24627-36 (1994).-   12. Luo, Y. et al. Side population cells from human melanoma tumors    reveal diverse mechanisms for chemoresistance. J Invest Dermatol    132, 2440-50.-   13. Wilson, B. J. et al. ABCB5 identifies a therapy-refractory tumor    cell population in colorectal cancer patients. Cancer Res 71,    5307-16 (2011).-   14. Hutcheson, D. A. & Kardon, G. Genetic manipulations reveal    dynamic cell and gene functions: Cre-ating a new view of myogenesis.    Cell Cycle 8, 3675-8 (2009).-   15. Lakso, M. et al. Efficient in vivo manipulation of mouse genomic    sequences at the zygote stage. Proc Natl Acad Sci USA 93, 5860-5    (1996).-   16. Meyer-Blazejewska, E. A. et al. From hair to cornea: toward the    therapeutic use of hair follicle-derived stem cells in the treatment    of limbal stem cell deficiency. Stem Cells 29, 57-66 (2011).-   17. Watanabe, K. et al. Human limbal epithelium contains side    population cells expressing the ATP-binding cassette transporter    ABCG2. FEBS Lett 565, 6-10 (2004).-   18. Frank, N. Y. et al. ABCB5-mediated doxorubicin transport and    chemoresistance in human malignant melanoma. Cancer Res 65, 4320-33    (2005).-   19. Cheung, S. T., Cheung, P. F., Cheng, C. K., Wong, N. C. &    Fan, S. T. Granulin-epithelin precursor and ATP-dependent binding    cassette (ABC)B5 regulate liver cancer cell chemoresistance.    Gastroenterology 140, 344-55 (2011).-   20. Yang, M. et al. Expression of ABCB5 gene in hematological    malignances and its significance. Leuk Lymphoma 53, 1211-5 (2012).-   21. Lehne, G. et al. Upregulation of stem cell genes in multidrug    resistant K562 leukemia cells. Leuk Res 33, 1379-85 (2009).-   22. Pellegrini, G. et al. Location and clonal analysis of stem cells    and their differentiated progeny in the human ocular surface. J Cell    Biol 145, 769-82 (1999).-   23. Meyer-Blazejewska, E. A. et al. Preservation of the limbal stem    cell phenotype by appropriate culture techniques. Invest Ophthalmol    Vis Sci 51, 765-74 (2010).-   24. Krulova, M. et al. A rapid separation of two distinct    populations of mouse corneal epithelial cells with limbal stem cell    characteristics by centrifugation on percoll gradient. Invest    Ophthalmol Vis Sci 49, 3903-8 (2008).-   25. Liu, P., Jenkins, N. A. & Copeland, N. G. A highly efficient    recombineering-based method for generating conditional knockout    mutations. Genome Res 13, 476-84 (2003).-   26. Rodriguez, C. I. et al. High-efficiency deleter mice show that    FLPe is an alternative to Cre-loxP. Nat Genet 25, 139-40 (2000).-   27. Frank, N. Y. et al. VEGFR-1 expressed by malignant    melanoma-initiating cells is required for tumor growth. Cancer Res    71, 1474-85 (2011).-   28. Frank, N. Y. et al. Regulation of myogenic progenitor    proliferation in human fetal skeletal muscle by BMP4 and its    antagonist Gremlin. J Cell Biol 175, 99-110 (2006).-   29. Pal-Ghosh, S., Pajoohesh-Ganji, A., Brown, M. & Stepp, M. A. A    mouse model for the study of recurrent corneal epithelial erosions:    alpha9beta1 integrin implicated in progression of the disease.    Invest Ophthalmol Vis Sci 45, 1775-88 (2004).-   30. Pellegrini, G. et al. The control of epidermal stem cells    (holoclones) in the treatment of massive full-thickness burns with    autologous keratinocytes cultured on fibrin. Transplantation 68,    868-79 (1999).-   31. Klocke, J. et al. Spontaneous bacterial keratitis in DC36    knockout mice. Invest Ophthalmol Vis Sci 52(1), 256-63 (2011)    (including Supplemental Appendix).-   32. Okita, K, et al. Generation of germline competent induced    pluripotent stem cells. Nature 448: 313-317 (2007).-   33. Stadfeld, M. and Hochedlinger, K. Induced pluripotency: history,    mechanisms, and applications, Genes and Development 24, 2239-2263    (2010).

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

1. A method of treating a subject having an ocular condition, comprisingadministering to the subject isolated ABCB5(+) stem cells in an amounteffective to regenerate ocular cells in the subject.
 2. The method ofclaim 1, wherein the ocular condition is a corneal disease.
 3. Themethod of claim 2, wherein the corneal condition is blindness due tolimbal stem cell deficiency (LSCD).
 4. The method of claim 1, whereinthe ocular condition is a retinal disease.
 5. The method of claim 4,wherein the retinal disease is macular degeneration.
 6. The method ofclaim 4, wherein the retinal disease is retinitis.
 7. The method ofclaim 1, wherein the ocular condition is an ocular wound.
 8. The methodof claim 1, wherein the isolated ABCB5(+) stem cells are administered asan ocular graft.
 9. The method of claim 1, wherein the isolated ABCB5(+)stem cells are allogeneic stem cells.
 10. The method of claim 1, whereinthe isolated ABCB5(+) stem cells are syngeneic stem cells.
 11. Themethod of claim 1, wherein the isolated ABCB5(+) stem cells are ABCB5(+)ocular stem cells.
 12. The method of claim 11, wherein the isolatedABCB5(+) ocular stem cells are ABCB5(+) limbal stem cells. 13-20.(canceled)
 21. A method of isolating limbal stem cells from a mixedpopulation of ocular cells, comprising: providing a mixed population ofocular cells; and isolating ABCB5(+) limbal stem cells from the mixedpopulation.
 22. The method of claim 21, wherein the ABCB5(+) limbal stemcells are ABCB5(+) human limbal stem cells.
 23. The method of claim 22,comprising contacting cells of the mixed population with an antibodythat selectively binds to human ABCB5. 24-30. (canceled)
 31. A method ofproducing an ocular graft for transplantation to a subject, comprisingseeding a substrate with isolated ABCB5(+) stem cells to produce theocular graft. 32-39. (canceled)
 40. An ocular graft enriched withisolated ABCB5(+) stem cells for transplantation in a subject. 41-46.(canceled)
 47. A method of promoting ocular cell regeneration,comprising identifying limbal stem cells as ABCB5(+) limbal stem cellsand administering to a subject in need thereof the ABCB5(+) limbal stemcells in an amount effective to promote ocular cell regeneration. 48.The method of claim 47, wherein the isolated ABCB5(+) limbal stem cellsare administered as an ocular graft.
 49. The method of claim 47, whereinthe isolated ABCB5(+) limbal stem cells are allogeneic stem cells.50-55. (canceled)
 56. An isolated preparation of limbal stem cellscharacterized by the expression of ABCB5 on their cell surface.